i

December 2014

U.S. Fish and Wildlife Service

Ecological Services

Raleigh, North Carolina

Pollutant Sensitivity of the Endangered Tar

River Spinymussel as Assessed by Single

Chemical and Effluent Toxicity Tests

ii

Preface

The U.S. Fish and Wildlife Service (USFWS), U.S. Geological Survey Columbia Environmental

Research Center (USGS CERC), North Carolina State University (NCSU) College of Veterinary

Medicine, and North Carolina Division of Water Resources (NCDWR) conducted an evaluation

of the habitat conditions for, and pollutant sensitivity of, the endangered Tar River spinymussel

(Elliptio steinstansana). The work was coordinated by the USFWS’s Raleigh Field Office and

was funded by the USFWS Division of Environmental Quality (study identifiers 4F42 and

200940001.1).

Toxicity tests were performed by the USGS CERC under the direction of Chris Ingersoll through

an intra-agency agreement with the USFWS. Other CERC scientists were involved in the toxicity

testing component of the project and included Ning Wang and James Kunz. Carol Hollenkamp

of NCDWR assisted with sample site selection and effluent collections. Mussel propagation was

coordinated by Jay Levine and Chris Eads of NCSU’s Aquatic Epidemiology and Conservation

Laboratory through an intra-agency agreement between the USFWS and the USGS North

Carolina Cooperative Fish and Wildlife Research Unit. Additional mussel propagation and

culture assistance was provided by Rachel Mair of the USFWS White Sulfur Springs National

Fish Hatchery. The overall project also included an inventory of nonpoint sources of pollution,

the results of which are provided elsewhere. Chris Mebane of USGS in Boise, Idaho performed

the biotic ligand model normalizations of copper toxicity test results. This report incorporates

comments received on a draft circulated for peer review. Peer reviews were provided by Greg

Cope of NCSU’s Department of Applied Ecology and Robert Bringolf of the University of

Georgia’s Warnell School of Forestry and Natural Resources; the co-authors thank them for their

time and expertise. The findings and conclusions in this report are those of the authors and do not

necessarily represent the views of the U.S. Fish and Wildlife Service.

Questions, comments, and suggestions related to this report can be directed to the USFWS at:

Tom Augspurger

U.S. Fish and Wildlife Service

P.O. Box 33726

Raleigh, North Carolina 27636-3726

tom_augspurger@fws.gov

Suggested citation: Augspurger TP, Wang N, Kunz JL, Ingersoll CG. 2014. Pollutant Sensitivity

of the Endangered Tar River Spinymussel as Assessed by Single Chemical and Effluent Toxicity

Tests. U.S. Fish and Wildlife Service, Ecological Services, Raleigh, NC.

Keywords: Tar River spinymussel, Elliptio steinstansana, Tar River, North Carolina, whole

effluent toxicity, 4F42, 200940001.1, NC-1, NC-7, NC-13

Cover photo: Juvenile Tar River spinymussels propagated at the North Carolina State University

Aquatic Epidemiology and Conservation Laboratory (photo by Chris Eads, Aquatic

Epidemiology and Conservation Laboratory, Raleigh, NC)

iii

Pollutant Sensitivity of the Endangered Tar River Spinymussel as Assessed by

Single Chemical and Effluent Toxicity Tests

Executive Summary: The federally endangered Tar River spinymussel (Elliptio

steinstansana) is endemic to the Tar River and Neuse River systems in North Carolina. The

extent to which water quality limits Tar River spinymussels’ recovery is important to establish,

and one aspect of that is understanding the species’ pollutant sensitivity. The primary objectives

of this study were to 1) develop captive propagation and culture methods for Tar River

spinymussels; 2) determine the pollutant sensitivity of captively propagated Tar River

spinymussels; 3) examine the utility of the non-endangered yellow lance (Elliptio lanceolata),

yellow lampmussel (Lampsilis cariosa) and notched rainbow (Villosa constricta) as surrogates

for the Tar River spinymussels’ chemical sensitivity; 4) develop a 7-d method for conducting

effluent toxicity tests starting with newly transformed mussels; 5) assess the toxicity of

municipal wastewater effluents discharged into the Tar River spinymussels’ current and historic

habitat; and, 6) evaluate the protection afforded by existing effluent toxicity test requirements.

From 2010 to 2012, propagation work for this project produced 23,714 juvenile Tar River

spinymussels, and this project demonstrated the utility of the white shiner (Luxilus albeolus) and

mountain redbelly dace (Phoxinus oreas) as highly effective host fish. In addition to supplying

Tar River spinymussels for the toxicity tests, thousands of juveniles were reared for potential

population augmentation / reintroduction efforts as a result of this project.

Field-collected effluents and laboratory-prepared mock effluents were used to conduct 7-d

toxicity tests with newly transformed juvenile mussels (notched rainbow and yellow lance) and

two commonly tested organisms (cladoceran, Ceriodaphnia dubia and fathead minnow,

Pimephales promelas). Acute (96-h) reference toxicant tests were also conducted with ammonia,

copper, or sodium chloride with four species of mussels, including Tar River spinymussels.

The Tar River spinymussel was between the median and bottom quartile of copper and ammonia

species sensitivity distributions for freshwater mussels based on 96-h toxicity tests. As such, the

Tar River spinymussel is a sensitive species among the mussels which, as a group, are known to

be sensitive to ammonia, chlorine, chloride, copper, nickel, lead, potassium, sulfate, and zinc.

While hazard is a function of sensitivity and exposure, the Tar River spinymussels’ sensitivity

indicates that pollutants are important factors to consider in its management.

The Tar River spinymussel, notched rainbow, and yellow lance were of similar (within a factor

of 2) sensitivity to ammonia and copper. The yellow lance had poor control survival in two of

two effluent tests and one of five acute tests. In contrast, notched rainbow performed well with

91 to 100% survival of controls in the seven effluent tests and 95 to 100% survival of controls in

the five acute tests conducted over three years. Although not as closely aligned with Tar River

spinymussel in taxonomy and distribution as is yellow lance, notched rainbow has the most

promise of the species we evaluated as a surrogate in Tar River spinymussel toxicity testing

based on availability, sensitivity, and control survival.

Three of five municipal wastewater treatment plant effluents were toxic to notched rainbow

mussels (used as a surrogate for the Tar River spinymussel) at concentrations approximately

iv

equal to, or less than the effluents’ instream waste concentration (the percent of stream flow as

effluent under the maximum permitted discharge during the estimated 7Q10 flow, or the 7-d low

flow with an expected recurrence interval of 10 years). Wastewater regulation using the instream

waste concentration is designed to be protective because facilities rarely discharge at maximum

permitted flow. However, there is the potential for toxicity to mussels at extreme low flow and

further evaluation in the lab and field is warranted.

Mock effluents were mixtures of ammonia, cadmium, copper, nickel, lead, and zinc. Mock

effluent 1 was expected to be toxic between the 12.5 and 25% concentrations as the 12.5%

concentration was mixed at the chronic water quality criteria of individual components. Mock

effluent 2 was expected to be toxic near the 12.5% concentration which was mixed at the

components’ individual thresholds of effect based on 28-d EC20s of other mussel species. The

IC20s for mussel length were 5.6 and 8.3% effluent indicating that no effect concentration

estimates based on previous mussel toxicity tests of individual compounds or based on water

quality criteria for individual compounds were similar, but those estimates are not protective of

the notched rainbow for the mixtures. The C. dubia were more sensitive than the mussels with

IC20s for reproduction of less than 3.2% for each effluent indicating adverse effects at

concentrations expected to be safe based on chronic water quality criteria for single chemicals.

In five of seven side-by-side comparisons (five wastewater treatment plant effluents and two

mock effluents), fathead minnow survival and reproduction were under-protective of mussel

endpoints. The fathead minnow does not consistently represent mussel sensitivity and would not

be an effective surrogate.

The C. dubia control survival was less than acceptable for three effluent tests and those results

were not evaluated further. In three of four side-by-side comparisons (two wastewater treatment

plant effluents and two mock effluents), C. dubia reproduction was protective of mussel

endpoints. Notched rainbow survival was more sensitive than C. dubia endpoints in one of the

two municipal effluents for which both species were tested. While C. dubia effluent toxicity

testing did not consistently protect mussel endpoints, the test should remain the main regulatory

tool for effluent evaluation in Tar River spinymussel habitat at this time because the limited

seasonal availability of these species of mussels would not support routine effluent testing. Tar

River spinymussel and notched rainbow can be tested directly when a mussel-specific test is

warranted based on the magnitude, composition, or location of an effluent. Both Tar River

spinymussel acute tests had acceptable control survival, and all seven effluent tests, conducted

over three years with different batches of notched rainbow had control survival ≥ 91% and good

growth. Hence the method can be used to monitor effluent toxicity in instances when a mussel

specific test is desired.

Growth (estimated as shell length) of mussels was a more sensitive endpoint compared to

survival with exposure to both of the mock effluents. In contrast, growth (estimated as shell

length) of mussels was not a sensitive endpoint in the exposures conducted with the wastewater

effluents. This is not surprising given the 7-d exposures. Length was used to measure growth

because of the difficulty in measuring dry weight increase in these small mussels. Future studies

should evaluate the utility of measuring dry weight of mussels in effluent testing, but

determining weight change of these young mussels would require very careful measurements.

v

Measuring dry weight would allow for estimating biomass of mussels (i.e., the total mass of

mussels in each replicate at the end of the exposures).

These are the first acute toxicity test results for ammonia and mussels in the genus Elliptio.

Using the same set of data quality objectives and same data synthesis procedures as those in the

USEPA’s 2013 revised ammonia water quality criteria document, Elliptio is the third most

sensitive of 70 genera of aquatic animals. As genus mean acute values close to the 5th

percentile

drive the criteria maximum concentrations (CMC) recommendations, the addition of the data for

Elliptio to the acute criteria dataset would lower the CMC by 11% (from 17 to 15 mg total

ammonia as nitrogen/L at pH 7.0 and 20°C) -- an illustration that even for a well-studied

pollutant like ammonia, available data for mussels have limitations in representing the U.S.’s

nearly 300 species of mussels in need of protection and that taxon-specific approaches to

deriving estimates of safe concentrations have merit in certain circumstances.

vi

CONTENTS

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

RESULTS / DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

MANAGEMENT IMPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Appendix

vii

TABLES Page

1. Wastewater treatment plant effluents evaluated, with flow and instream waste

concentrations (IWC). Results in the report are presented without attribution to

any particular facility. 3

2. Summary of conditions for conducting toxicity tests with juvenile notched

rainbow (Villosa constricta) mussels. 5

3. Summary of test conditions for conducting chronic effluent tests with the

cladoceran (Ceriodaphnia dubia). 6

4. Summary of test conditions for conducting toxicity tests with fathead minnow

(Pimephales promelas). 7

5. Mock effluent 1 for 7-d test with Ceriodaphnia dubia, fathead minnow

(Pimephales promelas) and the notched rainbow (Villosa constricta) freshwater

mussel in diluted well water. The 12.5% dilution was set at the USEPA criteria

continuous concentration at hardness 100 mg/L, DOC 0.5 mg/L, pH 8.3, and 20

or 22oC. 9

6. Mock effluent 2 for 7-d test with Ceriodaphnia dubia, fathead minnow

(Pimephales promelas) and the notched rainbow (Villosa constricta) mussel

in diluted well water. The 12.5% dilution (except for lead in 25% dilution due

to low solubility) was set close to the EC20 or geometric mean of the NOEC

and LOEC for fatmucket (Lampsilis siliquoidea) and rainbow (Villosa iris) in

previous 28-d water-only tests at hardness 100 mg/L, DOC 0.5 mg/L, pH 8.3,

and 20 or 22oC. 10

7. Summary of test conditions for conducting 4-day acute copper, ammonia, and

sodium chloride reference toxicant tests with juvenile Tar River spinymussel

(Elliptio steinstansana), yellow lance (Elliptio lanceolata), notched rainbow

(Villosa constricta), yellow lampmussel (Lampsilis cariosa), fathead minnows

(Pimephales promelas) and cladocerans (Ceriodaphnia dubia). 12

8. Responses of cladoceran (Ceriodaphnia dubia), fathead minnow

(Pimephales promelas), and notched rainbow (Villosa constricta) in 7-d

exposures to five permitted effluent samples. 14

9. Responses of cladoceran (Ceriodaphnia dubia), fathead minnow

(Pimephales promelas), and notched rainbow (Villosa constricta) in 7-d

exposures to two mock effluents. 16

viii

TABLES (continued) Page

10. EC50s (95% confidence interval) in acute 4-d reference toxicant tests

with the endangered Tar River spinymussel (Elliptio steinstansana) and three

potential surrogate mussel species (notched rainbow, Villosa constricta; yellow

lance, Elliptio lanceolata; and yellow lampmussel, Lampsilis cariosa) in ASTM

hard water. 17

11. Ranked freshwater mussel species mean acute values (SMAV) for ammonia

sensitivity. 18

12. Ranked freshwater mussel species mean acute values (SMAV) for copper

sensitivity. 19

13. Effluent toxicity tests comparing the sensitivity of freshwater mussels to

standard toxicity test organisms. 23

FIGURES

1. Juvenile Tar River spinymussels propagated at NCSU’s Aquatic Epidemiology

and Conservation Laboratory 1

1

Pollutant Sensitivity of the Endangered Tar River Spinymussel as Assessed by

Single Chemical and Effluent Toxicity Tests

Introduction

The federally endangered Tar River spinymussel (Elliptio steinstansana; Figure 1) is one of the

most imperiled species in North America. One of only three known freshwater mussel species

with spines in the world, the species is considered critically imperiled globally (G1 ranking by

NatureServe 2013). Listed as endangered by the U.S Fish and Wildlife Service (USFWS) in

1985, the species is known from just five locations in North Carolina. The Tar River spinymussel

recovery plan notes the role of water quality in its decline, stating that “degradation resulting

from siltation and the runoff and discharge of agricultural, municipal, and industrial pollutants

appear to be major factors in the reduction of the species’ range” (USFWS 1992).

3) Validate the utility of the non-endangered co-occurring and congeneric yellow lance (Elliptio

lanceolata), yellow lampmussel (Lampsilis cariosa), and notched rainbow (Villosa constricta) as

surrogates for the Tar River spinymussels’ chemical sensitivity;

4) Develop a 7-d method for conducting effluent toxicity tests with newly transformed mussels;

5) Assess the toxicity of municipal wastewater effluents discharged into the Tar River

spinymussels’ current and historic habitat; and,

6) Evaluate protection afforded to mussels by standard 7-d whole effluent toxicity tests (USEPA

2002) by conducting side-by-side effluent tests with mussels, the cladoceran (Ceriodaphnia dubia)

and fathead minnow (Pimephales promelas).

The approach of testing captively reared rare species has been successfully applied in the

conservation of other threatened and endangered mussels and fishes (Hamilton 1995, Buhl and

Figure 1. Juvenile Tar River spinymussels propagated at the

Aquatic Epidemiology and Conservation Laboratory (photo

by Chris Eads, NC State University)

To assist recovery efforts, the USFWS

coordinated an evaluation of the pollutant

sensitivity of the Tar River spinymussel. With

the assistance of the U.S. Geological Survey

Columbia Environmental Research Center

(USGS CERC), North Carolina State

University (NCSU) College of Veterinary

Medicine, and North Carolina Division of

Water Resources (NCDWR), this study was

initiated with the following objectives:

1) Develop captive propagation and culture

methods for Tar River spinymussels;

2) Determine the pollutant sensitivity of

captively propagated Tar River spinymussels;

2

Hamilton 1996, Keller and Augspurger 2005, Dwyer et al. 2005a, b, Besser et al. 2005, 2012,

Hewitt et al. 2006, Wang et al. 2007b). This project expands on that model and productive

partnerships (Augspurger et al. 1999, Noguchi et al. 2007). This report presents the results of the

captive propagation and testing of Tar River spinymussel and surrogate species including testing

methods, results, and an interpretation of the findings.

Methods

Overall design

The initial approach for the project was to test captively propagated Tar River spinymussels and

non-endangered yellow lance side-by-side with cladocerans and fathead minnows -- two species

commonly used to estimate acute and chronic toxicity of effluents and receiving waters (USEPA

2002). All species were to be tested with five field-collected effluents from North Carolina in 7-

d, static-renewal laboratory toxicity tests. While thousands of Tar River spinymussels were

eventually produced by project partners (NCSU 2013), there were none available in the first two

years of the project when effluents were collected for testing. The project was hence separated

into two phases: 1) an assessment of the sensitivity of mussels (notched rainbow and yellow

lance), cladocerans and fathead minnows to the five field-collected effluents (in 2010 and 2011)

or to two laboratory-prepared mock effluents (in 2012) in short-term 7-d exposures (to determine

the toxicity of the effluents to mussels and the relative sensitivity of the mussels to typical

effluent toxicity testing organisms), and 2) an assessment of the pollutant sensitivity of the Tar

River spinymussel relative to three other mussel species (yellow lance, yellow lampmussel, and

notched rainbow) in acute 96-h reference toxicant tests with ammonia, copper, or sodium

chloride. Overall objectives were still met, albeit indirectly.

Participating facilities and effluent collection

The NCDWR Aquatic Toxicology Unit coordinated a review of all the National Pollutant

Discharge Elimination System (NPDES) permitted wastewater treatment plant (WWTP)

discharges into occupied and historic habitat for the Tar River spinymussel. Facility type,

effluent composition, discharge volume, receiving stream, dilution at low flow, proximity to Tar

River spinymussel occurrences, compliance history with State toxicity testing requirements, and

other data were reviewed for effluent prioritization. Five facilities were selected for the study,

and NCDWR coordinated their voluntary participation (Table 1).

Two effluents were evaluated June 14-23, 2010 and three effluents were evaluated June 6-15,

2011. At each facility, effluent samples were collected directly into new 4-L certified clean

CUBITAINERS® containers following North Carolina Whole Effluent Toxicity Testing

procedures (NCDENR 1998). A 24-h composite of 20 liters of effluent was collected on Tuesday

(initiated Monday), chilled immediately, maintained at 1 to 4°C, and shipped overnight to the

USGS Columbia Environmental Research Center (USGS CERC) on Tuesday. Effluents were

received by USGS on Wednesday when testing commenced. A second 24-h effluent composite

sample was collected at each facility on Thursday (initiated Wednesday) and shipped to USGS

CERC on Thursday for effluent renewal in test chambers. Tests ended the following Wednesday.

3

Table 1. Wastewater treatment plant effluents evaluated and their maximum permitted flow (in

million gallons per day, MGD). The instream waste concentration (IWC) is the percent of the

stream flow that is comprised by a particular facility’s maximum permitted effluent during 7Q10

flows (the predicted seven-day low flow with an average recurrence interval of once every ten

years). Test results in this report are presented without attribution to particular facilities.

Facility County Receiving

stream

Permitted

flow (MGD) IWC

(%)

Kenly WWTP Johnston Little River 0.63 22

Louisburg Water Reclamation Facility Franklin Tar River 1.37 13

Tar River Regional WWTP Nash Tar River 21 35

Tarboro WWTP Edgecombe Tar River 5 8

Warren County WWTP Warren Fishing Creek 2 76

Test organism culture

Neonate C. dubia (less than 24-h old and all within 8 h of the same age) were cultured at the

USGS CERC in control water (dilution water) prepared by: 1) diluting well water with deionized

water to a hardness about 100 mg/L as CaCO3, pH 8.2, and dissolved organic carbon about 0.5

mg/L (Wang et al. 2011b) and 2) preparing ASTM reconstituted hard water (160-180 mg/L as

CaCO3, pH 8.3, and dissolved organic carbon about 0.3 mg/L; ASTM 2013a). Fathead minnows

(<24-h old) were obtained from Aquatic Bio Systems Inc., Fort Collins, Colorado.

Juvenile notched rainbow were supplied by White Sulfur Spring National Fish Hatchery. In April

each year, gravid female mussels to produce juvenile notched rainbow were collected from Johns

Creek, Maggie, Virginia. Fish used to produce juvenile mussels were sculpin (Cottus sp.) and

were collected from Howards Creek, Greenbrier County, West Virginia. Fish were held in

AHAB-style systems with sieves to collect the juvenile mussels once they metamorphosed from

the fish. Fish were held in these systems at about 20⁰C and about 2 to 3 weeks later, juveniles

excysted from the fish. Juveniles were then counted and placed in Barnhart Bucket (Barnhart

2006) juvenile mussel culture systems until they were shipped 1 to 2 d later to the USGS CERC

for toxicity testing.

Juvenile Tar River spinymussel, yellow lance, and yellow lampmussel were supplied by the

NCSU Aquatic Epidemiology and Conservation Lab (AECL) in Raleigh, North Carolina (NCSU

2013). Tar River spinymussel brood stock was collected from Little Fishing Creek in the Tar

River Basin over multiple years. Tar River spinymussels were maintained at the Marion

Conservation Aquaculture Center (Marion, North Carolina) where they spawned and became

gravid. Gravid females were transported to the AECL for production of juveniles by host fish

transformation of glochidia. Between 2010-2012, the AECL infected 818 fish across 42

infestation events, producing 23,714 juvenile Tar River spinymussels. White shiner (Luxilus

albeolus) was the primary host used for all propagation efforts, but the mountain redbelly dace

(Phoxinus oreas) was identified as another highly effective host. For all species, the newly

transformed juveniles obtained from 1 to 3 collection days during the peak drop-off of juveniles

from the host fish were shipped overnight to USGS CERC for testing (NCSU 2013).

4

WWTP effluent toxicity testing

Once received by USGS CERC, juvenile mussels were acclimated to control water (dilution

water) and test temperature (25°C) for 2 d before the start of the toxicity testing (ASTM 2013b).

The juvenile mussels were fed an algal mixture (Nannochloropsis concentrate and Shellfish Diet,

Reed Mariculture, Campbell, California; Wang et al. 2007a) during the acclimation period twice

daily in the morning and afternoon. Algal density in the acclimation containers was about 5 to 10

nl cell volume/ml after each feeding. At the beginning of each WWTP effluent test (Table 2), ten

juvenile mussels exhibiting foot movement were impartially transferred into each of four

replicate 300-ml glass beakers containing about 200 ml of 100%, 50%, 25%, 12.5%, 6.25% test

effluent, and dilution water (control water). In addition, about 20 juveniles were also impartially

sampled and preserved in 80% ethanol for initial length measurement (see initial lengths in

Tables A2 and A4). About 75% water in each replicate was removed and renewed daily. Mussels

were fed 2 ml of the algal mixture once daily after water renewal.

Survival of juvenile mussels was determined at the end of the test. Juvenile mussels were

classified as alive if they exhibited foot movement within a 5 min observation period using a

dissecting microscope (ASTM 2013b). The test acceptability criterion was > 80% control

survival. Surviving mussels at the end of the tests were preserved for shell length determinations.

The maximum shell length of each surviving mussel was measured to the nearest 0.001 mm

using a digitizing system with video micrometer software (Image Caliper, Resolution

Technology, Dublin, Ohio).

Neonate C. dubia were assigned impartially to test chambers by placing one organism in one of

ten replicates per effluent concentration (Table 3). Test chambers were placed in a water bath at

25ºC. Each day before water renewal, each first-generation C. dubia was recorded as alive or

dead (immobility as lack of movement within 5 seconds in response to gentle prodding) and was

transferred to a new test chamber containing fresh exposure water. The number of young

released from females over each 24-h period was recorded. The C. dubia were fed 0.1 ml each

yeast-cerophyll-trout chow (YCT; 1800 mg/L stock solution) and algal (Pseudokirchnerella

subcapitata) suspension (3.0 to 3.5 X 107 cell/ml) per chamber daily. Exposures were conducted

until 60% of the control cladocerans produced three broods (a 7-d exposure). The test

acceptability criterion was ≥80% control survival and ≥15 young/female in controls.

Fathead minnows were acclimated to control water and test temperature (25°C) for 24 h before

testing (Table 4). The fish were fed newly hatched (less than 24-h old) brine shrimp (Artemia)

nauplii twice daily at a rate of adding 1 ml of a concentrated suspension of the nauplii into 2 L of

water during the acclimation period. At the beginning of a test, ten fish (<48-h old) were

impartially transferred into each of four replicate 500-ml glass beakers containing about 250 ml

of water. About 80% of the water was renewed daily. The fish were fed 0.15 ml of a

concentrated suspension of less than 24-h-old brine shrimp nauplii twice daily on test day 0 to 6.

Sufficient numbers of nauplii were provided to assure that some nauplii remain alive in the test

chamber for several hours after each feeding. Fish survival was determined at the end of the test.

Surviving fish per replicate were dried at 60ºC for 24 h for dry weight measurement. Biomass

was then determined as total dry weight of surviving fish in a replicate. The test acceptability

criterion was ≥80% control survival and average dry weight per surviving individual in control

chambers equals or exceeds 0.25 mg.

5

Table 2. Summary of conditions for conducting effluent toxicity tests with juvenile mussels

(notched rainbow, Villosa constricta; yellow lance, Elliptio lanceolata) in basic accordance with

ASTM (2013a, b) and USEPA (2002).

Test species: Notched rainbow and yellow lance

Test chemicals: Five field-collected effluent samples from North Carolina

Test type: Static renewal

Test Duration: 7 d

Temperature: 251C

Light quality: Ambient laboratory light

Light intensity: 200 lux

Photoperiod: 16L:8D

Test chamber size: 300 ml

Test solution volume: 200 ml

Renewal of solution: Daily (about 75% replacement of water)

Age of test organism: About 7 days after transformation

Number of organisms per

test chamber: 10

Number replicate chambers

per concentration: 4

Feeding: 2 ml of algal mixture once daily after water renewal

Chamber cleaning: None

Aeration: None

Dilution water: Diluted well water (100 mg/L as CaCO3)

Dilution factor: 0.5

Test concentration: 0, 6.25, 12.5, 25, 50, 100% effluent

Chemical residues: Major cations and anions in 100% effluents at start of test

Water quality: pH, conductivity, hardness, alkalinity measured in the control,

medium and high exposure concentrations on Days 0 and 7;

dissolved oxygen measured every other day.

Endpoint: Survival and growth (shell length)

Test acceptability criterion: ≥80% control survival (no test acceptability criteria have been

established for 7-d test with newly transformed mussels)

6

Table 3. Summary of test conditions for conducting chronic effluent tests with the cladoceran

(Ceriodaphnia dubia) in basic accordance with USEPA (2002) and ASTM (2013c).

Test species: Ceriodaphnia dubia

Test chemicals: Five field-collected effluent samples from North Carolina

Test type: Static renewal

Test Duration: 7 d, when 60% of control animals produce 3 broods

Temperature: 251C

Light quality: Ambient laboratory light

Light intensity: 200 lux

Photoperiod: 16L:8D

Test chamber size: 30 ml (disposable polystyrene cup)

Test solution volume: 15 ml

Renewal of solution: Daily

Age of test organism: <24 h (within 8 hours of age)

No. organisms per

test chamber: 1

No. replicate chambers

per concentration: 10

Feeding: 0.1 ml each yeast-cerophyll-trout chow (YCT; 1800 mg/L stock

solution) and algal (Raphidocelis subcapitata) suspension (3.0 to

3.5 X 107 cell/ml) per chamber daily

Chamber cleaning: New plastic cups daily

Aeration: None

Dilution water: Diluted well water (100 mg/L as CaCO3)

Dilution factor: 0.5

Test concentration: 0, 6.25, 12.5, 25, 50, 100% effluent

Chemical residues: Major cations and anions in 100% effluents at start of test

Water quality: pH, conductivity, hardness, alkalinity measured in the control,

medium and high exposure concentrations on days 0 and 7;

dissolved oxygen measured every other day.

Endpoint: Survival and reproduction

Test acceptability criterion: ≥80% control survival and ≥15 young/female in controls and

≥60% of surviving control females had three broods.

7

Table 4. Summary of test conditions for conducting toxicity tests with fathead minnow

(Pimephales promelas) in basic accordance with USEPA (2002).

Test species: Fathead minnow

Test chemicals: Five field-collected effluent samples from North Carolina

Test type: Static renewal

Test Duration: 7 d

Temperature: 251C

Light quality: Ambient laboratory light

Light intensity: 200 lux

Photoperiod: 16L:8D

Test chamber size: 500 ml

Test solution volume: 250 ml

Renewal of solution: Daily (about 80% replacement of water)

Age of test organism: <48 h

Number of organisms per

test chamber: 10

Number replicate chambers

per concentration: 4

Feeding: On days 0-6, feed 0.15 ml of concentrated suspension of

24-h-old brine shrimp nauplii twice daily (early morning and

afternoon after water renewal).

Chamber cleaning: Siphon daily, immediately before water renewal

Aeration: None, unless dissolved oxygen concentration <4.0 mg/L

Dilution water: Diluted well water (100 mg/L as CaCO3)

Dilution factor: 0.5

Test concentration: 0, 6.25, 12.5, 25, 50, 100% effluent

Chemical residues: Major cations and anions in 100% effluents at start of test

Water quality: pH, conductivity, hardness, alkalinity measured in the control,

medium and high exposure concentrations on days 0 and 7;

dissolved oxygen measured every other day.

Endpoint: Survival and biomass (based on dry weight)

Test acceptability criterion: ≥80% control survival and average dry weight per surviving

organism in control chambers > 0.25 mg/individual.

8

Mock effluent preparation

The sensitivity of test organisms to mock effluents was evaluated in 2012. Mock effluent 1 was a

mixture of ammonia (NH4Cl), cadmium (CdCl2), copper (CuSO4), nickel (NiCl2), lead (PbNO3),

and zinc (ZnCl2). The USEPA chronic ambient water quality criteria for these chemicals were

selected as the concentrations of equitoxic proportion for each toxicant (Table 5). The 12.5%

dilution was set at the criteria continuous concentration (red text in Table 5). Mock effluent 2

was a mixture of the same chemicals as in mock effluent 1, but the concentrations of equitoxic

proportion were selected based on EC20s or chronic values (geometric mean of NOEC and

LOEC) for mussels (Table 6). The 12.5% dilution was set at the EC20 or chronic value for

mussels (red text in Table 6) except the 25% dilution was used for lead due to low solubility.

Because mussels and C. dubia are generally more sensitive to metals than fathead minnows, the

mussels and C. dubia were tested in six dilutions of 0, 3.2, 6.25, 12.5, 25, and 50% of each mock

effluent while fathead minnows were tested in six dilutions of 0, 6.25, 12.5, 25, 50, and 100%.

Mock effluent toxicity testing

Mock effluent tests were conducted with notched rainbow mussels, cladocerans and fathead

minnows using protocols described previously for the WWTP effluent tests (Tables 2 to 4) with

the following modifications. The control water (dilution water) in mock effluent tests was

reconstituted ASTM hard water (160-180 mg/L as CaCO3; ASTM 2013a) and light intensity was

500 lux. To improve the process of daily water renewal and recovery of mussels at the end of the

test, newly designed mussel exposure units were used in the mock effluent tests. The exposure

units consisted of a 160-ml inner chamber and a 200-ml outer beaker (modified from the

exposure unit described in Miao et al. 2010). The inner chamber was a glass tube with stainless-

steel screen (120-µm opening) at the bottom. Each of four replicate 200-ml glass beakers

contained about 150 ml of water. Feeding was 1 ml of the algal mixture twice daily after water

renewal. Water in each replicate exposure unit was removed and renewed (100%) daily. Testing

the mussels in these exposure units made it easier to renew water daily during the exposures and

to recover the mussels at the end of the exposures. Survival of juvenile mussels was determined

at the end of the test. Surviving mussels were preserved in ethanol (80%) for subsequent shell

length measurement.

9

Conc. (µg/L) Control 3.13% 6.25% 12.5% 25% 50% 100%

Ammonia Control 250 500 1000 2000 4000 8000

Cadmium Control 0.06 0.13 0.25 0.5 1 2

Copper Control 0.38 0.75 1.5 3 6 12

Nickel Control 13 26 52 104 208 416

Lead Control 1.25 1.25 2.5 5 10 10

Zinc Control 30 60 120 240 480 960

Acute toxic unit to mussels, fatmucket or paper pondshell a

Ammonia Control 0.04 0.08 0.15 0.31 0.62 1.23

Cadmium Control 0.00 0.00 0.01 0.02 0.04 0.08

Copper Control 0.02 0.03 0.06 0.12 0.24 0.48

Nickel Control 0.05 0.10 0.21 0.41 0.83 1.65

Lead Control 0.00 0.00 0.00 0.01 0.01 0.01

Zinc Control 0.10 0.19 0.39 0.77 1.55 3.10

Sum 0.21 0.41 0.82 1.64 3.28 6.55

EC50 (µg/L) Reference

Ammonia 6500 Fatmucket (Lampsilis siliquoidea); Wang et al. 2008

Cadmium 26 Fatmucket; Wang et al. 2010

Copper 25 Fatmucket; Wang et al. 2009

Nickel 252 Paper pondshell (Utterbackia imbecillis); Keller et al. 1991

Lead 670 Fatmucket; Wang et al. 2010

Zinc 310 Fatmucket; Wang et al. 2010

a The toxic units are determined based on hardness or BLM-normalized EC50s from

previous 96-h tests with newly transformed mussels:

Table 5. Mock effluent 1 for 7-d test with Ceriodaphnia dubia, fathead minnow

(Pimephales promelas) and the notched rainbow (Villosa constricta) freshwater

mussel in diluted well water (hardness 100 mg/L as CaCO3, dissolved organic carbon

0.5 mg/L). The 12.5% dilution (red text) was set at the USEPA criteria continuous

concentration at hardness 100 mg/L, DOC 0.5 mg/L, pH 8.3, and 20 or 22oC.

10

Conc. (µg/L) Control 3.13% 6.25% 12.5% 25% 50% 100%

Ammonia Control 100 200 400 800 1600 3200

Cadmium Control 2 4 8 16 32 64

Copper Control 2 4 8 16 32 64

Nickel Control 11.5 23 46 92 184 368

Lead Control 6 11 22 44 88 176

Zinc Control 31 62 124 248 496 992

Acute toxic unit to fatmucket or paper pondshell a

Ammonia Control 0.02 0.03 0.06 0.12 0.25 0.49

Cadmium Control 0.08 0.15 0.31 0.62 1.23 2.46

Copper Control 0.08 0.16 0.32 0.64 1.28 2.56

Nickel Control 0.05 0.09 0.18 0.37 0.73 1.46

Lead Control 0.01 0.02 0.03 0.07 0.13 0.26

Zinc Control 0.10 0.20 0.40 0.80 1.60 3.20

Sum 0.33 0.65 1.30 2.61 5.22 10.44

EC50 (µg/L) Reference

Ammonia 6500 Fatmucket (Lampsilis siliquoidea); Wang et al. 2008

Cadmium 26 Fatmucket; Wang et al. 2010

Copper 25 Fatmucket; Wang et al. 2009

Nickel 252 Paper pondshell (Utterbackia imbecillis); Keller et al. 1991

Lead 670 Fatmucket; Wang et al. 2010

Zinc 310 Fatmucket; Wang et al. 2010

Table 6. Mock effluent 2 for 7-d test with Ceriodaphnia dubia, fathead minnow

(Pimephales promelas) and notched rainbow (Villosa constricta) mussel in diluted well

water (hardness 100 mg/L as CaCO3, dissolved organic carbon 0.5 mg/L). The 12.5%

dilution (red text) was set close to the EC20 or geometric mean of the NOEC

and LOEC for fatmucket (Lampsilis siliquoidea) and rainbow (Villosa iris) mussels in

previous 28-d water-only tests at hardness 100 mg/L, DOC 0.5 mg/L, pH 8.3, and 20

or 22oC (except for lead in 25% dilution due to low solubility).

a The toxic units are determined based on hardness or BLM-normalized EC50s from

previous 96-hour tests with newly transformed mussels:

11

Reference toxicant tests

Juvenile mussels were acclimated to the control water (the ASTM reconstituted hard water;

ASTM 2013a) and test temperature (20°C) for 2 d before testing. The juvenile mussels were fed

algal mixture (Wang et al. 2007a) at a rate of 2 ml of the algal mixture into 200 ml of water twice

daily during the acclimation period. Test conditions are summarized in Table 7. At the beginning

of the 4-d static-renewal reference toxicant test with copper or sodium chloride, five mussels

were impartially transferred into each of four replicate 50-ml glass beakers containing about 30

ml of test solution. Mussels were not fed during the exposure. Test solution was renewed at 48 h.

Ammonia reference toxicity tests were conducted in a flow-through diluter system to maintain

constant ammonia concentrations during the 4-d exposures. Ten mussels were impartially

transferred into each exposure unit which consisted of a 160-ml inner chamber and a 300-ml

outer beaker (Miao et al. 2010). The inner chamber was a glass tube with stainless-steel screen

(120-µm opening) at the bottom. The diluter system delivered about 120 ml of test solution into

each inner chamber once every hour. Mussels were not fed during the exposure.

Survival of mussels was determined at the end of the tests. The test acceptability was ≥90%

control survival in the reference toxicant tests.

Water quality and chemical analysis

Dissolved oxygen, pH, conductivity, hardness, alkalinity, and total ammonia nitrogen were

measured using standard methods (Eaton et al. 2005) on composite water samples collected from

the replicates in the control, medium, and high concentrations at the beginning and end of each

test. Dissolved oxygen was measured every other day on composite water samples collected

before daily water renewal. Water samples (filtered through a 0.45-µm pore size membrane) for

major cations (calcium, potassium, magnesium, and sodium) and major anions (chloride and

sulfate) were collected in the 100% effluents at the start of WWTP effluent exposures. The

cation samples were stabilized within 24 h by adding 16 M nitric acid to each sample at a volume

proportion of 1:100 (1% v/v). Major cations were analyzed by Laboratory and Environmental

Testing (Columbia, Missouri), using inductively coupled plasma atomic emission spectroscopy

(ICPAES) according to the U.S. Environmental Protection Agency method 200.7 (USEPA

1994). Major anions were analyzed at the USGS CERC using ion chromatography in basic

accordance with USEPA (2007a) method 9056A.

For the mock effluent exposures, water samples (20 ml) for analyses of the five metals were

collected in each exposure concentration at the beginning and end of the test with a

polypropylene syringe, filtered through a 0.45-µm pore size polyethersulfone membrane into a

polyethylene bottle, and stabilized within 24 h by adding 16 M nitric acid to each sample at a

volume proportion of 1:100. Concentrations of the five metals were determined by inductively

coupled plasma-mass spectrometry (ICP-MS), (PE/SCIEX ELAN DRCe, PerkinElmer, Norwalk,

Connecticut) in accordance with U.S. Environmental Protection Agency method 6020A (USEPA

2007a). Ammonia was also analyzed daily in each of the exposure concentrations. The

concentration of ammonia was determined with an Orion ammonia electrode and Orion EA940

meter (Thermo Electron, Beverly, Massachusetts).

12

Table 7. Summary of test conditions for conducting 4-day acute copper, ammonia, and sodium

chloride reference toxicant tests with juvenile Tar River spinymussel (Elliptio steinstansana),

yellow lance (Elliptio lanceolata), notched rainbow (Villosa constricta), yellow lampmussel

(Lampsilis cariosa), fathead minnows (Pimephales promelas) and cladocerans (Ceriodaphnia

dubia) in basic accordance with ASTM (2013a, b).

Test chemicals: CuSO4, NH4Cl, NaCl

Test type: Static renewal (copper, sodium chloride), flow through (ammonia)

Test Duration: 96 h

Temperature: 20C

Light quality: Ambient laboratory light

Light intensity: 200 lux

Photoperiod: 16L:8D

Test chamber size: Static renewal: 50 ml for mussel and cladoceran,

500 ml for minnow;

Flow through: 300-ml outer beaker with 160-ml inner chamber

(Miao et al 2010)

Test solution volume: Static renewal: 30 ml for mussel and cladoceran,

250 ml for minnow;

Flow through: 200 ml (outer beaker) and 100 ml (inner chamber)

Renewal of solution: Static renewal: After 48 h

Flow through: Additional 120-ml to each beaker once every 4 h

Age of test organism: Mussel: about 7 after transformation

Cladoceran: <24 h

Fathead minnow: 48 h

No. organisms per

test chamber: 5 or 10 (mussel), 1 (cladoceran), or 10 (minnow)

No. replicate chambers

per concentration: 4 (mussels),10 (cladoceran), or 2 (minnow)

Feeding: No feeding (except 0.2 ml Artemia nauplii concentrate 2 h before

water renewal at 48 h for minnow)

Chamber cleaning: None

Aeration: None

Dilution water: Reconstituted ASTM hard water (160-180 mg/L as CaCO3)

Dilution factor: 0.5

Test concentration: 0, 1, 2, 4, 8, 16 g NaCl/L

0, 1.0, 2.0, 4.0, 8.0 and 16 mg total ammonia nitrogen/L

0, 6.25, 12.5, 25, 50, 100 µg copper/L

Chemical residues: Ammonia in each concentration measured at least every other day.

Water samples for copper and sodium chloride analysis collected

at the beginning and the end of test

Water quality: Dissolved oxygen, pH, conductivity, hardness, and alkalinity

determined at the control, medium, and high exposure

concentrations at the beginning and the end of test

Endpoint: Survival

Test acceptability criterion: ≥90% control survival

13

The meter was calibrated before measuring samples with 1.0 and 10 mg N/L calibration

standards. The method detection limit was 0.03 mg N/L. Water samples for dissolved organic

carbon (DOC) analysis were collected from the control beakers and shipped overnight in a cooler

with ice packs to Huffman Laboratories (Golden, Colorado). Water samples were filtered at 0.45

µm before analysis. The Huffman laboratory used a TOC Analyzer (OI Analytical Model 700)

following persulfate-ultraviolet oxidation with infrared detection (method 5310C; Eaton et al.

2005) and reported a method detection limit of 0.05 mg C/L.

For the reference toxicant tests, ammonia was measured at each exposure concentration at the

beginning and the end of tests, and copper was measured at each concentration at the beginning

of the test. The NaCl concentrations were not measured. Salinity and conductivity were

measured at the beginning and the end of each test to confirm the target NaCl concentrations.

Data analysis

No-observed-effect concentration (NOEC), lowest-observed-effect concentration (LOEC), and

20% inhibition concentration (IC20) for survival, length (mussels), biomass (minnows), or

reproduction (C. dubia) in the short-term 7-d effluent tests, and 50% effect concentration (EC50)

in the acute reference toxicant tests were estimated using TOXSTAT software (version 3.5,

Western EcoSystems Technology, Cheyenne, Wyoming) following the methods outlined in

USEPA (2002). The EC50s in the reference toxicant tests for copper or ammonia were calculated

based on measured concentrations, and the EC50s in the reference toxicant tests for NaCl were

calculated based on nominal concentrations.

Results and Discussion

Whole effluent toxicity tests

Table 8 summarizes the WWTP effluent toxicity test results; the appendix Tables A1 to A4

provide individual test results and supporting water chemistry. Control survival of notched

rainbow and fathead minnow was acceptable in all effluent tests (notched rainbow control

survival ranged from 91 to 100%, and fathead minnow control survival ranged from 85 to

100%). The C. dubia control survival was 90% and control reproduction was 21 and 22 young

per female in effluents 1 and 2 (tested in 2010 with the same batch of C. dubia) but was only 30,

60, and 100% in effluents 3, 4, and 5 (tested in 2011 with the same batch of C. dubia). The C.

dubia control reproduction was also low in the three effluents tested in 2011, ranging from 9 to

15. Therefore, C. dubia results for effluents 3, 4, and 5 are not included in Table 8. In addition,

yellow lance was tested in effluents 1 and 2 in 2010. The control survival was 51% (Table A2)

and did not meet the test acceptability requirement of ≥80% control survival (Table 2).

Therefore, the effect concentrations for yellow lance were not included in Table 8.

Based on IC20s, four of the five effluents were toxic to at least one test species (all except for

effluent 5) (Table 8). In effluent 1, C. dubia reproduction was a more sensitive endpoint than any

endpoints of the notched rainbow or fathead minnows (Table 8). However, in effluent 2 notched

rainbow survival and growth were both more sensitive endpoints than any endpoints of C. dubia

and fathead minnows. In effluents 3 and 4, there were no acceptable test results for C. dubia for

comparison to mussels, but the mussels were always more sensitive than fathead minnows.

14

The IC20s were used to evaluate effluent toxicity and to compare sensitivity of the test species.

While informative in a weight of evidence approach, the NOEC and LOEC are generated by

post-analysis analysis of variance (ANOVA) multiple comparison tests and have important

limitations (e.g., NOEC and LOEC can only take values of a tested concentration, failure to

reject a null hypothesis of no difference does not mean there was no effect because a biologically

significant effect may occur and not be detected by the ANOVA). For example, in the toxicity

test with effluent 1, the NOEC was 100% but the IC20 was <6.25% and the biological effect

observed was 23 to 37% reduction in growth (length) relative to the control. Others have

observed between 10 and 34% effects occurring at the reported NOEC when test data are re-

evaluated by a regression-derived estimate rather than hypothesis testing (Crane and Newman

2000).

Mussels are known to be sensitive to potassium, sulfate, and chloride (Soucek 2006, USEPA

2010, Gillis 2011, Wang et al. 2012, 2013, Ivey et al. 2013) and relatively tolerant of nitrate

Table 8. Effect concentrations of cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and

notched rainbow (Villosa constricta) in 7-d exposures to five permitted effluent samples. The no-observed-effect

concentration (NOEC), the lowest-observed-effect concentration (LOEC), and 20% inhibition concentrations (IC20)

with 95% confidence interval (CI) are presented for each endpoint. See Table A2 and Table A4 for additional detail.

Cladoceran

Fathead minnow

Notched rainbow

Treatment Survival Reproduction Survival Biomass Survival Length

Effluent 1

NOEC (%) 100 100 100 100

100 100

LOEC (%) >100 >100

>100 >100

>100 >100

IC20 (CI; %) >100 <6.25 >100 >100 >100 >100

Effluent 2

NOEC (%) 100 100 100 50 50 25

LOEC (%) >100 >100

>100 100

100 50

IC20 (CI; %) >100 >100 >100 >100 24 (12-67) >100

Effluent 3

NOEC (%) NRa NR 100 100 50 50

LOEC (%) NR NR >100 >100 100 >50

IC20 (CI; %) NR NR >100 >100 <6.25 >100

Effluent 4

NOEC (%) NR NR 100 100 12.5 12.5

LOEC (%) NR NR >100 >100 25 >12.5

IC20 (CI; %) NR NR >100 >100 69 (23-120) >100

Effluent 5

NOEC (%) NR NR 100 100 100 100

LOEC (%) NR NR >100 >100 >100 >100

IC20 (CI; %) NR NR >100 >100 >100 >100 a Not reported because of low control survival.

15

(USEPA 2010, Soucek and Dickinson 2012) which were all among the anions and cations

analyzed in the whole effluents (Tables A1 and A3). Potassium 96-h EC50s for six mussel

species range from about 31 to 52 mg/L (Wang et al. 2013); an approximate no to low effect

concentration of 15 mg/L (from taking the low EC50 and dividing by two) is not exceeded in any

effluent but is approached by effluents 3, 4 and 5 which had potassium concentrations between

10.5 and 13.9 mg/L (Tables A1 and A3). There were no concentrations of the other major ions in

the effluents exceeding those known to be harmful to mussels based on the references cited

above. Mussels are also known to be sensitive to ammonia (USEPA 2013), and effluent 2

contained 0.91 to 3.41 mg/L total ammonia as nitrogen (TAN) over the course of the 7-d

exposures (Table A1). While no follow-up toxicity identification evaluation was performed for

the effluents, it is plausible that ammonia was the source of toxicity. Ammonia concentrations

exceeded those known to be lethal to freshwater mussels (Wang et al. 2007b, 2008) and

exceeded acute and chronic water quality criteria for ammonia (normalized to pH 7 and 20oC;

USEPA 2013).

The percent of the stream flow that is comprised by a particular facility’s maximum permitted

effluent during 7Q10 flows (the predicted seven-day low flow with an average recurrence

interval of once every ten years) is known as the instream waste concentration (IWC). The

IWC’s for effluents 2, 3, and 4 were approximately equal to, or greater than concentrations that

impacted mussel survival. While results are presented without attribution to particular facilities,

effluents 2, 3, and 4 were toxic to mussels at concentrations that potentially may occur in the

environment during situations of maximum permitted discharge under low flow conditions.

Mock effluent toxicity tests

Table 9 summarizes the mock effluent toxicity test results; appendix Tables B1 to B3 provide the

individual test results and supporting water chemistry. The measured concentrations of toxicants

(ammonia and the five metals) typically ranged from 80 to 120% of the nominal concentrations

(Table B1). Control survival for each of three test species was 100% and met test acceptability

criteria for each mock effluent, and reproduction of C. dubia and weight of fathead minnows also

met test acceptability criteria. The two mock effluents were toxic to all test species, and the

sublethal endpoints (C. dubia reproduction, fathead minnow biomass, and notched rainbow

mussel length) were generally more sensitive than the lethal endpoint (Table 9). Based on the

IC20s for the sublethal endpoints, the species sensitivity to the two mock effluents was C. dubia

> notched rainbow > fathead minnow. However, the LOECs based on mussel length were lower

than the LOECs based on C. dubia reproduction (Table 9).

The 12.5% dilution in mock effluent 1 consisted of toxicants mixed at the USEPA chronic water

quality criteria (those in effect in 2012) for the hardness, pH, and DOC of the dilution water and

test temperature. Accordingly, the threshold for toxicity in mock effluent 1 was expected to be

between the 12.5 and 25% concentrations. However, mock effluent 1 was more toxic to mussels

and C. dubia than expected; the IC20 for C. dubia survival (7%), C. dubia reproduction (<3.1%)

and mussel length (5.6%) indicated effects at concentrations less than those expected to be safe

based on 2012 chronic water quality criteria of individual compounds.

16

The 12.5% treatment in mock effluent 2 consisted of toxicants mixed near their individual

thresholds of effect based on EC20s of other mussel species in 28-d exposures. Accordingly, the

threshold for toxicity to mussels in mock effluent 2 was expected to be near the 12.5%

concentration. However, the IC20 for mussel length (8.3%) indicates an effect at concentrations

less than those expected to be safe based on published mussel chronic toxicity test results of

individual compounds used in the effluents.

Based on mortality, the effluents exhibited similar toxicity to notched rainbow with survival

IC20s at about 25%. The IC20s for mussel length were also similar (5.6 and 8.3%, but with

overlapping confidence intervals). This indicates that no effect concentration estimates based on

previous mussel toxicity tests of individual compounds or based on water quality criteria for

individual compounds were similar, but those estimates are not protective of the notched rainbow

for the mixture of metals and ammonia in these tests.

Reference toxicant tests

The EC50s for acute 4-d reference toxicant (sodium chloride, copper, or ammonia) tests with the

four mussel species are provided in Table 10 (individual test results and supporting water

chemistry are in the appendix Tables C1 to C8). Control survival in all acute tests was >90%,

except one ammonia test with yellow lance (76% control survival, the EC50 for which is not

included in Table 10). The three table entries with more than one value (notched rainbow copper

and sodium chloride, and yellow lance copper) are results from reference toxicant tests

conducted in different years of the project and generally show consistency of results between

years. The notched rainbow 3-fold difference in copper sensitivity between years is within the

range of historical intra-species, intra-lab test variation (e.g., Wang et al. 2007b, c), but at the

high end of the range for intra-lab test variation. Tar River spinymussel, yellow lance and

notched rainbow were of similar sensitivity to ammonia, with overlapping EC50 95% confidence

intervals (Table 10). Yellow lance was the most sensitive species to copper among the four tested

species, and was also more sensitive than notched rainbow to sodium chloride (Table 10).

Table 9. Effect concentrations of cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and

notched rainbow (Villosa constricta) in 7-d exposures to two mock effluents. The no-observed-effect concentration

(NOEC), the lowest-observed-effect concentration (LOEC), and 20% inhibition concentrations (IC20) with 95%

confidence interval (CI) are presented for each endpoint. See Table B3 for additional detail.

Cladoceran

Fathead minnow

Notched rainbow

Treatment Survival Reproduction Survival Biomass Survival Length

Mock Effluent 1

NOEC (%) 6.25 3.125

25 25

25 <3.125

LOEC (%) 12.5 6.25

50 50

50 3.125

IC20 (CI; %) 7.0 (4.3-7.8) <3.1 34 (21-41) 33 (20-40) 25 (15-28) 5.6 (4.2-10)

Mock Effluent 2

NOEC (%) 6.25 3.125 25 12.5 25 <3.125

LOEC (%) 12.5 6.25

50 25

50 3.125

IC20 (CI; %) 6.9 (4.3-7.5) <3.2 29 (19-36) 20 (16-28) 26 (21-29) 8.3 (5.7-11)

17

Table 10. EC50s (95% confidence interval) in acute 4-d reference toxicant tests with the endangered Tar

River spinymussel (Elliptio steinstansana) and three potential surrogate mussel species (notched rainbow,

Villosa constricta; yellow lance, Elliptio lanceolata; and yellow lampmussel, Lampsilis cariosa) in

ASTM reconstituted hard water (ASTM 2013a).

Tar River

spinymussel

Notched rainbow Yellow lance Yellow

lampmussel

Copper

(µg Cu/L)

26 (22-31) 12 (9.6-15)a

39 (32-48)b

16 (13-20)a

16 (13-19)b

46 (32-68)

Total ammonia

(mg N/L)

3.6 (3.1-4.3) 3.5 (2.9-4.3) 3.1 (2.6-3.7) 8.2 (7.2-9.2)

Sodium chloride

(g/L)

NTd 3.9 (3.3-4.7)

c

4.1 (3.5-4.8)a

2.1 (1.8-2.5) NT

a test conducted in 2011

b test conducted in 2012

c test conducted 2010

d Not tested.

To compare relative species sensitivity with other mussels tested in previous studies, species

mean acute values (SMAVs) were calculated and ranked in Table 11 for ammonia and Table 12

for copper. An SMAV is the geometric mean of 24 to 96-h EC50s from all acceptable tests for a

species. Because no toxicity tests were conducted with sodium chloride and the endangered Tar

River spinymussel, no species sensitivity comparison with other mussels was conducted. The

aquatic toxicity of ammonia varies by temperature and pH, and the toxicity of copper is

influenced by several water quality characteristics, including pH, concentrations of cations and

anions, and dissolved organic carbon. We used the ammonia toxicity temperature-pH

normalization equations of the revised USEPA water quality criteria for ammonia (USEPA

2013) to normalize acute values for ammonia SMAV calculations. The Tar River spinymussel,

notched rainbow, and yellow lance, were of similar sensitivity within or near the bottom quartile

of the ammonia sensitivity distribution for mussels (Table 11), indicating notched rainbow and

yellow lance would be suitable surrogates for Tar River spinymussel ammonia sensitivity.

However, the yellow lampmussel was more tolerant than these species and would not appear to

be a good surrogate for the Tar River spinymussel based on ammonia sensitivity.

These are the first acute toxicity test results for ammonia and mussels in the genus Elliptio.

Using the same set of data quality objectives and same data synthesis procedures as those in the

revised ammonia water quality criteria document (USEPA 2013), the genus Elliptio can be added

to the data used to derive the criteria. Elliptio is the third most sensitive of 70 genera of aquatic

animals in the expanded dataset with a genus mean acute value (GMAV, the geometric mean of

the SMAVs by genus) of 31.05 mg/L total ammonia as N at pH 7 and 20oC (there is some

uncertainty about the phylogenetic classification of Tar River spinymussel, but because the

SMAV for the yellow lance is less than that of the Tar River spinymussel, the GMAV for

Elliptio would still be the third most sensitive if Tar River spinymussel was assigned to another

genus). As genus mean acute values close to the 5th

percentile drive the criteria maximum

concentration (CMC) recommendations, the addition of the data for Elliptio to the acute criteria

18

Table 11. Ranked ammonia genus mean acute values (GMAVs) with associated species mean acute

values (SMAVs). An SMAV is the geometric mean of 24 to 96-h EC50s from all acceptable tests and a

GMAV is the geometric mean of the SMAVs by genus. Data are from Table 3 of the revised ambient

water quality criteria for ammonia (USEPA 2013) with newly tested North Carolina mussel species from

this study added (in red). All values are normalized to mg-N/L at pH 7 and 20°C (USEPA 2013). The

2013 and 1999 criteria maximum concentrations and final acute values are provided for reference.

GMAV (mg N/L) Species SMAV (mg N/L)

109.0 Dwarf wedgemussel, Alasmidonta heterodon 109.0

109.0 Pink papershell, Potamilus ohiensis 109.0

72 1999 FAVa

71.25 Mucket, Actinonaias ligamentina 63.89

Pheasantshell, Actinonaias pectorosa 79.46

70.73 Giant floater mussel, Pyganodon grandis 70.73

50.01 Pink mucket, Lampsilis abrupta 26.03

Plain pocketbook, Lampsilis cardium 50.51

Yellow lampmussel, Lampsilis cariosa 76.22

Wavy-rayed lampmussel, Lampsilis fasciola 48.11

Higgin's eye, Lampsilis higginsii 41.90

Neosho mucket, Lampsilis rafinesqueana 69.97

Fatmucket, Lampsilis siliquoidea 55.42

47.40 Atlantic pigtoe, Fusconaia masoni 47.40

46.93 Pondshell mussel, Utterbackia imbecillis 46.93

36 1999 CMCb

33.52 2013 FAVc

33.37 Notched rainbow, Villosa constricta 32.53

Rainbow mussel, Villosa iris 34.23

31.14 Oyster mussel, Epioblasma capsaeformis 31.14

31.05 Yellow lance, Elliptio lanceolata 28.81

Tar River spinymussel, Elliptio steinstansana 33.46

23.41 Green floater, Lasmigona subviridus 23.41

23.12 Ellipse, Venustaconcha ellipsiformis 23.12

17 2013 CMCd

a Final Acute Value, Salmonids absent (USEPA 1999).

b Criterion Maximum Concentration, Salmonids absent - estimate of the highest concentration to

which an aquatic community can be exposed briefly without resulting in an unacceptable effect

(USEPA 1999). The CMC is the FAV divided by two. c Final Acute Value (USEPA 2013).

d Criterion Maximum Concentration (USEPA 2013).

19

dataset would lower the CMC by 11%. It is also interesting that adding the data for two

additional species of Lampsilis and Villosa, genera already represented in the dataset, changed

the value of their GMAVs but had little influence on their ranks (i.e., the new SMAVs were

similar to those already available for other species in those genera). While it is also possible to

look at the Elliptio GMAV as similar (within a factor of two) of the of those already in the

database and, therefore, of not much value, it should serve as a reminder that even for a well-

studied pollutant like ammonia, the data for 17 mussel species have limitations in representing

the U.S.’s nearly 300 species of mussels in need of protection and that alternate approaches to

deriving estimates of safe concentrations have merit in certain circumstances (Augspurger 2013).

Biotic ligand models have been developed to enable mechanistic modeling of copper

bioavailability and acute toxicity as a function of metal speciation and the protective effects of

competing cations. We used the biotic ligand model normalization equations of HydroQual Inc.

(2007) to normalize acute values for copper SMAV calculations. The yellow lance was the

second most sensitive mussel species to copper; the Tar River spinymussel and notched rainbow

were of similar sensitivity in the median of the mussel species sensitivity distribution for copper

(Table 12). However, the yellow lampmussel was more tolerant than these species. Similar to the

conclusion based on the comparisons of ammonia sensitivity, the notched rainbow and yellow

lance, but not the yellow lampmussel, would be suitable surrogates for the Tar River spinymussel

based on the difference in copper sensitivity.

Table 12. Ranked freshwater mussel genus mean acute values (GMAVs) for copper with

associated species mean acute values (SMAVs). An SMAV is the geometric mean of 24 to 96-h

EC50s from all acceptable tests and a GMAV is the geometric mean of the SMAVs by genus. Data

are biotic ligand model (BLM)-normalized from Table S3 of Wang et al. 2009 with species from

this study added (in red). The USEPA (2007b) criteria maximum concentration (CMC) and final

acute value (FAV) are provided for reference.

GMAV (μg/L) Species SMAV (μg/L)

55.3 Green floater, Lasmigona subviridis 55.3

15.8 Pondshell, Utterbackia imbecillis 15.8

12.7 Yellow lampmussel, Lampsilis cariosa 21.1

Neosho mucket, Lampsilis rafinesqueana 15.1

Wavy-rayed lampmussel, Lampsilis fasciola 9.43

Fatmucket, Lampsilis siliquoidea 8.58

11.5 Rainbow, Villosa iris 13.0

Notched rainbow, Villosa constricta 10.1

10.3 Tar River spinymussel, Elliptio steinstansana 12.9

Yellow lance, Elliptio lanceolata 8.24

9.90 Scaleshell, Leptodea leptodon 9.90

4.79 Oyster mussel, Epioblasma capsaeformis 4.79

FAV

a 4.7

CMC

b 2.3

a Final Acute Value (USEPA 2007b).

b Criterion Maximum Concentration (USEPA 2007b).

20

Management Implications

Freshwater mussels are one of North America’s most imperiled faunal groups (Lydeard et al.

2006). Thirty-five species of freshwater mussels are extinct (Turgeon et al. 1998) and 86 species

are federally listed as threatened or endangered. Widespread and chronic impacts such as water

pollution and physical habitat alteration are considered the most important current impairments

(Richter et al. 1997, Strayer et al. 2004). Toxic effects are reasonable hypotheses to test as

limiting factors for mussels because aspects of their life history make them vulnerable to

degraded water or sediment quality. Juvenile and adult mussels are benthic suspension feeders

exposed to pollutants in surface water, sediment, and pore water and through ingestion of filtered

particles with sorbed contaminants (Cope et al. 2008). Chemical aspects of water quality have

been historically cited as detrimental to mussels; associations between mussel decline and

pollutant sources or impaired water quality have been documented for decades (Fuller 1974).

The Tar River spinymussel recovery plan speaks to water quality overtly, and this project has

helped address components of the action plan (USFWS 2009) and recovery plan (USFWS 1992),

particularly Recovery Task 2.2 - Identify and eliminate current and future threats to the species’

survival, including water quality and habitat degradation. Each of the study objectives outlined

on page 1 is revisited here with regard to management implications.

1) The project further developed captive propagation and culture methods for Tar River

spinymussels.

The project expanded knowledge of Tar River spinymussel life history and advanced its

propagation and culture (NCSU 2013). Fish host identification is a high priority action in the Tar

spinymussel recovery plan (USFWS 1992), and this project demonstrated the utility of the white

shiner (Luxilus albeolus) and mountain redbelly dace (Phoxinus oreas) as highly effective host

fish (NCSU 2013). The white shiner is commonly collected from the same streams as Tar River

spinymussel and was the most effective host fish in the laboratory transformations. From 2010 to

2012, Tar River spinymussel propagation work for this project produced 23,714 juvenile Tar

River spinymussels. In 2013, a total of 57 individuals from the 2010-year class remained alive at

the North Carolina Wildlife Resources Commission hatchery in Marion, North Carolina and had

grown to 30-37 mm in length. There were 1,074 individuals produced from the 2011-year class

at the Marion facility that were approximately 12 to 22 mm in length. As of spring 2013, there

were thousands of surviving juveniles from the 2012-year class ranging in length from 700-1500

µm at NCSU’s Aquatic Epidemiology and Conservation Laboratory. Those are being reared for

potential population augmentation or reintroduction efforts as a result of this project (NCSU

2013).

2) The project determined that Tar River spinymussels appear to be sensitive to contaminants.

Freshwater mussels are among the most sensitive forms of aquatic life to toxicity from ammonia,

chlorine, chloride, copper, nickel, lead, potassium, sulfate, and zinc (Augspurger et al. 2003,

Soucek 2006; Wang et al. 2007a, b, c, 2008, 2009, 2010, 2011a, b, 2012, 2013; March et al.

2007; Besser et al. 2011, 2013; Gillis 2011; Ivey et al. 2013) which are common pollutants of

surface waters. We tested the acute sensitivity of the Tar River spinymussel to ammonia and

copper (Table 11 and 12); the Tar River spinymussel was between the median and bottom

21

quartile of the species sensitivity distributions for mussels. As such, the Tar River spinymussel is

a sensitive species among the mussels which, as a group, are known to be sensitive to ammonia

and copper. It is reasonable to expect that the species may also be sensitive to other pollutants

known to be of concern to mussels, including chloride, chlorine, lead, nickel, potassium, sulfate,

and zinc (Wang et al. 2013). While hazard is a function of sensitivity and exposure, the species’

sensitivity indicates pollutants are important factors to consider in management of the Tar River

spinymussel.

Three effluents were toxic to notched rainbow mussels (used as a surrogate for the Tar River

spinymussel) at concentrations approximately equal to or less than the effluents’ instream waste

concentration, or IWC – concentrations that potentially may occur in the environment during

situations of maximum permitted discharge under low flow conditions. Wastewater regulation

using the IWC is designed to be protective in this scenario that is unlikely to occur because

facilities rarely discharge at maximum permitted flow. However, there is the potential for

toxicity to mussels at extreme low flow from some of the facilities’ maximum permitted

discharge, and some existing effluents should be further evaluated in the lab and field.

Mock effluents were expected to be toxic between the 12.5 and 25% concentrations as the 12.5%

concentration was mixed at the chronic water quality criteria of individual components (mock

effluent 1) or their individual thresholds of effect based on EC20s of other mussel species in 28-d

exposures (mock effluent 2). The IC20s for mussel length were 5.6 and 8.3% effluent which are

about 2-times lower than concentrations expected to be safe. This indicates that no effect

concentration estimates based on previous mussel toxicity tests of individual compounds or

based on water quality criteria for individual compounds were similar, but those estimates are

not protective of the notched rainbow for the mixture of metals and ammonia in these tests.

3) The non-endangered co-occurring and congeneric yellow lance and the notched rainbow have

promise as surrogates for Tar River spinymussels’ chemical sensitivity.

The Tar River spinymussel, notched rainbow, and yellow lance were of similar (within a factor

of 2) sensitivity to ammonia and copper. The yellow lampmussel was more tolerant than these

species and would not appear to be a good surrogate based on the difference in chemical

sensitivity. The yellow lance had poor control survival in the effluent tests (appendix Table A2).

While one bad test performance does not make a bad surrogate, notched rainbow performed well

with 91 to 100% survival of experimental controls in all tests conducted over the three year

study. The notched rainbow is also native to Tar and Neuse River drainages (as well as the

Catawba, Pee Dee, Cape Fear, Roanoke and Chowan river basins). Although not as closely

aligned with Tar River spinymussel in its taxonomy and distribution as is yellow lance, notched

rainbow has the most promise of the species that we evaluated as a surrogate in Tar River

spinymussel toxicity testing based on its availability, sensitivity, and control survival.

4) In seven side-by-side comparisons (five WWTP effluents and two mock effluents), fathead

minnow survival and reproduction were frequently under-protective of mussel endpoints.

The objective of a whole effluent toxicity test is to determine whether an effluent concentration

equal to the wastewater’s maximum permitted concentration in the receiving stream during low

stream flow conditions has significant detrimental impact upon reproduction and survival, or

22

growth of test organisms (NCDWQ 2010a, b). Two WWTP effluents were not toxic to mussels

or fathead minnows. In the other three WWTP effluents and both mock effluents, the mussels

were more sensitive than fathead minnows. The fathead minnow does not consistently represent

mussel sensitivity.

5) There were no C. dubia to Tar River spinymussel effluent test comparisons and insufficient C.

dubia to notched rainbow effluent test comparisons to definitively determine the protectiveness of

the C. dubia test for freshwater mussels. In side-by-side comparisons (two WWTP effluents and

two mock effluents), C. dubia reproduction was protective of mussel endpoints in three of four

tests. While C. dubia effluent toxicity testing did not consistently protect mussel endpoints, the

test should remain the main regulatory tool for effluent evaluation in Tar River spinymussel

habitat at this time. Tar River spinymussel and notched rainbow can be tested directly when a

mussel-specific test is warranted based on the magnitude, composition, or location of an effluent.

Notched rainbow survival and growth (determined as shell length) were more sensitive than C.

dubia endpoints in one of the two municipal effluents for which both species were tested. While

mussel growth was a sensitive endpoint, it has limitations. For example, the toxicant may

selectively kill small mussels and increase the mean length of surviving mussels, or no

meaningful length data may be obtainable in high exposure concentrations when all mussels die

(Table B3). Also, the lab reported that it was very difficult to obtain dry weight for the newly

transformed juveniles (and, therefore, not able to calculate biomass). Future studies should

evaluate the utility of measuring dry weight of mussels in effluent testing, but weight

determination of these young mussels would require very careful measurements. Measuring dry

weight would allow for estimating biomass of mussels (i.e., the total mass of mussels in each

replicate at the end of the exposures). Also, producing newly transformed juvenile mussels for

effluent testing is not routine, with seasonal constraints on the availability of juveniles for most

species making it more difficult to obtain test organisms on a regular basis compared to C. dubia.

Considering these challenges to estimating sublethal effect concentrations in effluent testing with

mussels and that the 7-d C. dubia chronic test was protective of mussel endpoints in three of four

tests, the C. dubia survival and reproduction test should remain the main regulatory tool for

effluent evaluation in Tar River spinymussel habitat at this time. Side-by-side tests of effluents

with C. dubia and Tar River spinymussels, as was originally envisioned, remains a research need

that may be accommodated as propagation methods and capacity improve.

There will be cases when a location, composition, or scale of a discharge makes the additional

effort to conduct a mussel toxicity test warranted. Just as notched rainbow were more sensitive

than C. dubia in one of our four side-by-side comparisons, others evaluating surrogates for

mussels in whole effluent toxicity testing note that C. dubia tests are typically, but not always

protective of mussels (Table 13). The successful propagation of Tar River spinymussels and

surrogates as part of this project (NCSU 2013) provides the opportunity for additional testing of

these species directly when a water quality issue merits that level of scrutiny.

23

Table 13. Effluent toxicity tests comparing the sensitivity of freshwater mussels to standard

toxicity test organisms including Ceriodaphnia dubia (CD) and fathead minnow (FHM).

Reference Species evaluated Type of effluent Results

This study Villosa constricta,

CD, FHM

Municipal wastewater Villosa constricta

more sensitive than

CD in 1 of 2 effluents.

Both species more

sensitive than FHM.

This study Villosa constricta,

CD, FHM

Mixtures of ammonia,

cadmium, copper,

nickel, lead, and zinc

CD and Villosa

constricta of similar

sensitivity. Both

species more sensitive

than FHM.

EPRI 2011 Anodonta imbecilis,

CD

Coal-fired power plant Anodonta imbecilis

and CD of

comparable sensitivity

McKinney et al. 1996 Anodonta imbecilis,

CD

Pulp and paper mill Anodonta imbecilis

substantially more

sensitive than CD

Masnado et al. 1995 Anodonta imbecilis,

CD, FHM

Synthetic mine effluent

(mixture of Cd, Cr, Cu,

Ni and Zn)

CD always more

sensitive than

Anodonta imbecilis

Keller 1993 Anodonta imbecilis,

CD, FM

Municipal wastewater CD more sensitive

than Anodonta

imbecilis

References

American Society for Testing and Materials. 2013a. Standard guide for conducting acute toxicity

tests on test materials with fishes, macroinvertebrates, and amphibians (ASTM E729-96 (2007)).

In Annual Book of ASTM Standards, Volume 11.06. ASTM, West Conshohocken, PA.

American Society for Testing and Materials. 2013b. Standard method for conducting laboratory

toxicity tests with freshwater mussels (E2455-06 (2013)). In Annual Book of ASTM Standards,

Volume 11.06. ASTM, West Conshohocken, PA.

24

American Society for Testing and Materials. 2013c. Standard guide for conducting three-brood,

renewal toxicity tests with Ceriodaphnia dubia (ASTM E1295-01 (2013)). In Annual Book of

ASTM Standards, Volume 11.06. ASTM, West Conshohocken, PA.

Augspurger T, Keller AE, Black MC, Cope WG, Dwyer FJ. 2003. Water quality guidance for

protection of freshwater mussels (Unionidae) from ammonia exposure. Environ Toxicol Chem

22: 2569-2575.

Augspurger T. 2013. Site-specific ammonia recommendation for the South Toe River’s (NC)

endangered freshwater mussels. Poster RP099, Society of Environmental Toxicology and

Chemistry annual meeting, November 21, Nashville, TN.

Augspurger T, Dwyer J, Fridell J. 1999. Fishing for answers. End Species Bull 24: 8-9.

Barnhart MC. 2006. Bucket of muckets: A compact recirculating system for rearing juvenile

freshwater mussels. Aquaculture 254: 227-233.

Besser JM, Brumbaugh WG, Kemble NE, Ivey CD, Kunz JL, Ingersoll CG, Rudel D. 2011.

Toxicity of nickel-spiked freshwater sediments to benthic invertebrates—Spiking methodology,

species sensitivity, and nickel bioavailability: U.S. Geological Survey Scientific Investigations

Report 2011–5225, 53 p. plus appendixes.

Besser JM, Brumbaugh WG, Papoulias DM, Ivey CD, Kunz JL, Annis M, Ingersoll CG. 2012.

Bioaccumulation and toxicity of selenium during a life-cycle exposure with desert pupfish

(Cyprinodon macularius): U.S. Geological Survey Scientific Investigations Report 2012–5033,

30 p. with appendixes (http://pubs.usgs.gov/sir/2012/5033/).

Besser JM, Brumbaugh WG, Ingersoll CG, Ivey CD, Kunz JL, Kemble NE, Schlekat CE,

Rogevich-Garman E. 2013. Chronic toxicity of nickel-spiked freshwater sediments: Variation in

toxicity among eight invertebrate taxa and eight sediments. Environ Toxicol Chem 32:2495-

2506.

Besser JM, Wang N, Dwyer FJ, Mayer FL Jr, Ingersoll CG. 2005. Assessing contaminant

sensitivity of endangered and threatened aquatic species: Part II. Chronic toxicity of copper and

pentachlorophenol to two endangered species and two surrogate species. Arch Environ Contam

Toxicol 48: 155-65.

Buhl KJ, Hamilton SJ. 1996. Toxicity of inorganic contaminants, individually and in

environmental mixtures, to three endangered fishes (Colorado squawfish, bonytail, and

razorback sucker). Arch Environ Contam Toxicol 30: 84-92.

Cope WG, Bringolf RB, Buchwalter DB, Newton TJ, Ingersoll CG, Wang N, Augspurger T,

Dwyer FJ, Barnhart MC, Neves RJ, Hammer E. 2008. Differential exposure, duration, and

sensitivity of unionoidean bivalve life stages to environmental contaminants. J North Am

Benthol Soc 27: 451-462.

25

Crane M, Newman MC. 2000. What level of effect is a no observed effect? Environ Toxicol

Chem19: 516-519.

Dwyer FJ, Mayer FL, Sappington LC, Buckler DR, Bridges CM, Greer IE, Hardesty DK, Henke

CE, Ingersoll CG, Kunz JL, Whites DW, Augspurger T, Mount DR, Hattala K, Neuderfer GN.

2005a. Assessing contaminant sensitivity of endangered and threatened aquatic species: Part 1.

Acute toxicity of five chemicals. Arch Environ Contam Toxicol 48: 143-154.

Dwyer FJ, Hardesty DK, Henke CE, Ingersoll CG, Whites DW, Augspurger T, Canfield TJ,

Mount DR, Mayer FL. 2005b. Assessing contaminant sensitivity of endangered and threatened

aquatic species: Part III. Effluent toxicity tests. Arch Environ Contam Toxicol 48: 174-183.

Eaton AD, Clesceri LS, Rice EW, and Greenberg AE. 2005. Standard Methods for the

Examination of Water and Wastewater, 21st ed.: Washington, D.C., American Public Health

Association, Water Environment Federation, American Water Works Association, 1,368 p.

EPRI. 2011. Characterization of toxicity of coal-fired power plant effluents to freshwater

mussels. 2011 Technical report 1021820, EPRI, Palo Alto, CA.

Fuller SLH. 1974. Clams and mussels (Mollusca: Bivalvia). In Hart CW Jr, Fuller SLH, eds,

Pollution Ecology of Freshwater Invertebrates. Academic, New York, NY, USA, pp 215-273.

Gillis PL. 2011. Assessing the toxicity of sodium chloride to the glochidia of freshwater mussels:

implications for salinization of surface waters. Environ Pollut 159:1702-1708.

Hamilton SJ. 1995. Hazard assessment of inorganics to three endangered fish in the Green River,

Utah. Ecotoxicol Environ Safety 30: 134-142.

Hewitt AH, Cope WG, Kwak TJ, Augspurger T, Lazaro PR, Shea D. 2006. Influence of water

quality and associated contaminants on survival and growth of the endangered Cape Fear shiner.

Environ Toxicol Chem 25: 2288-2298.

HydroQual, Inc. 2007. The Biotic Ligand Model Windows Interface, Version 2.2.3: User’s

Guide and Reference Manual, HydroQual, Inc., Mahwah, NJ.

Ivey CD, Consbrock RA, Kunz JL, Ingersoll CG, Wang N. Hammer E, Bauer CR, Augspurger

T, Raimondo S, Barnhart C, Brumbaugh BG. 2013. Sensitivity of freshwater mussels at two life

stages to acute or chronic effects of sodium chloride or potassium chloride. Poster MP031,

Society of Environmental Toxicology and Chemistry annual meeting, November 18, Nashville,

TN.

Keller AE. 1993. Acute toxicity of several pesticides, organic compounds, and wastewater

effluent to the freshwater mussels Anodonta imbecilis, Ceriodaphnia dubia, and Pimephales

promelas. Bull Environ Contam Toxicol 51:696-702.

Keller AE, Augspurger T. 2005. Toxicity of fluoride to the endangered unionid mussel,

Alasmidonta raveneliana, and surrogate species. Bull Environ Contam Toxicol 74: 242-249.

26

Lydeard C, Cowie RH, Ponder WF, Bogan AE, Bouchet P, Clark SA, Cummings KS, Frest TJ,

Gargominy O, Herbert DG, Hershler R, Perez KE, Roth B, Seddon M, Strong EE, Thompson

FG. 2004. The global decline of nonmarine mollusks. BioScience 54:321-330.

March FA, Dwyer FJ, Augspurger T, Ingersoll CG, Wang N, Mebane CA. 2007. An evaluation

of freshwater mussel toxicity data in the derivation of water quality guidance and standards for

copper. Environ Toxicol Chem 26: 2066-2074.

Masnado RG, Geis SW, Sonzogni WC. 1995. Comparative acute toxicity of a synthetic mine

effluent to Ceriodaphnia dubia, larval fathead minnow and the freshwater mussel Anodonta

imbecilis. Environ Toxicol Chem 14: 1913-1920.

McKinney AD, Wade DC. 1996. Comparative response of Ceriodaphnia dubia and juvenile

Anodonta imbecillis to pulp and paper mill effluents discharged to the Tennessee River and its

tributaries. Environ Toxicol Chem 15: 514-517.

Miao J, Barnhart MC, Brunson EL, Hardesty DK, Ingersoll CG, Wang N. 2010. An evaluation of

the influence of substrate on the response of juvenile freshwater mussels (Fatmucket, Lampsilis

siliquoidea) in acute water exposure to ammonia. Environ Toxicol Chem 29:2112-2116.

NatureServe. 2013. NatureServe Explorer: An online encyclopedia of life [web application].

Version 7.1. NatureServe, Arlington, Virginia. Available http://www.natureserve.org/explorer.

(Accessed: August 8, 2013 ).

North Carolina Division of Water Quality. 2010a. North Carolina Ceriodaphnia Chronic Whole

Effluent Toxicity Procedure. Version 3.0, Raleigh, NC.

North Carolina Division of Water Quality. 2010b. North Carolina Phase II Chronic Whole

Effluent Toxicity Test Procedure. Version 3.0, Raleigh, NC.

North Carolina Department of Environment and Natural Resources (NCDENR). 1998. North

Carolina Phase II, Chronic Whole Effluent Toxicity Test Procedure. North Carolina Department

of Environment and Natural Resources, Division of Water Quality, Water Quality Section,

Raleigh, NC.

North Carolina State University. 2013. Final report for fish host identification, culture, and

propagation of the Tar Spinymussel and Yellow Lance, two rare endemic mussels of the North

Carolina Piedmont. NCSU, College of Veterinary Medicine, Aquatic Epidemiology and

Conservation Laboratory, Raleigh, NC. 41 pp.

Noguchi G, Augspurger T, Dwyer J. 2007. Clearing the water for mussels. End Species Bull 32:

14-15.

Richter BD, Braun DP, Mendelson MA, Master LL. 1997. Threats to imperiled freshwater fauna.

Conserv Biol 11:1081-1093.

27

Soucek DJ. 2006. Effects of water quality on acute and chronic toxicity of sulfate to freshwater

bivalves, Ceriodaphnia dubia and Hyalella azteca. Final Report. Illinois Natural History Survey

Champaig, IL.

Soucek DJ, Dickinson A. 2012. Acute toxicity of nitrate and nitrite to sensitive freshwater

insects, mollusks, and a crustacean. Arch Environ Contam Toxicol 62:233-242.

Strayer DL, Downing JA, Haag WR, King TL, Layzer JB, Newton TJ, Nichols SJ. 2004.

Changing perspectives on pearly mussels, North America’s most imperiled animals. BioScience

54:429-439.

Turgeon DD, Quinn JF, Bogan AE, Coan EV, Hochberg FG, Lyons WG, Mikkelsen PM, Neves

RJ, Roper CFE, Rosenberg G, Roth B, Scheltema A, Thompson FG, Vecchione M, Williams JD.

1998. Common and Scientific Names of Aquatic Invertebrates from the United States and

Canada: Mollusks, 2nd ed. Special Publication 26. American Fisheries Society, Bethesda, MD.

U.S. Environmental Protection Agency. 1994. Method 200.7 Determination of metals and trace

elements in water and wastes by inductively coupled plasma-atomic emission spectrometry.

Revision 4.4. Environmental Monitoring Systems Laboratory, Office of Research and

Development, Cincinnati, OH.

U.S. Environmental Protection Agency. 1999. 1999 Update of ambient water quality criteria for

ammonia. EPA-822-R-99-014. Office of Water, Office of Science and Technology

Washington, D.C.

U.S. Environmental Protection Agency. 2002. Short-term methods for estimating the chronic

toxicity of effluents and receiving water to freshwater organisms. EPA-821-R-02-013,

Washington, DC.

U.S. Environmental Protection Agency. 2007a. Method 6020A: Inductively Coupled Plasma-

Mass Spectrometry, in: SW-846 Online: Test Methods for Evaluating Solid Waste,

Physical/Chemical Methods.

U.S. Environmental Protection Agency. 2007b. Aquatic life ambient freshwater quality criteria—

Copper, 2007 revision. EPA-822-R-07-001. Office of Water, Washington, DC.

United States Environmental Protection Agency. 2010. Final report on acute and

chronic toxicity of nitrate, nitrite, boron, manganese, fluoride, chloride and sulfate to several

aquatic animal species. EPA 905-R-10-002. USEPA, Region 5, Chicago, IL.

U.S. Environmental Protection Agency. 2013. Aquatic life ambient water quality criteria for

ammonia – freshwater. EPA 822-R-13-001. Office of Water, Office of Science and Technology,

Washington, DC.

U.S. Fish and Wildlife Service. 1992. Revised Tar Spinymussel Recovery Plan. Atlanta, GA.

28

U.S. Fish and Wildlife Service. 2009. Tar River Spinymussel Recovery Action Plan. U.S. Fish

and Wildlife Service, Asheville, North Carolina Ecological Services Field Office.

http://www.fws.gov/ecos/ajax/docs/action_plans/doc3109.pdf

Wang N, Ingersoll CG, Greer IE, Hardesty DK, Ivey CD, Kunz JL, Dwyer FJ, Roberts AD,

Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007a. Chronic toxicity of copper and

ammonia to juvenile freshwater mussels (Unionidae). Environ Toxicol Chem 26:2048-2056.

Wang N, Ingersoll CG, Hardesty DK, Ivey CD, Kunz JL, May TW, Dwyer FJ, Roberts AD,

Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007b. Acute toxicity of copper, ammonia,

and chlorine to glochidia and juveniles of freshwater mussels (Unionidae). Environ Toxicol

Chem 26:2036-2047.

Wang N, Augspurger T, Barnhart MC, Bidwell JR, Cope WG, Dwyer FJ, Geis S, Greer IE,

Ingersoll CG, Kane CM, May TW, Neves RJ, Newton TJ, Roberts AD, Whites DW. 2007c.

Intra- and inter-laboratory variability in acute toxicity tests with glochidia and juveniles of

freshwater mussels (Unionidae). Environ Toxicol Chem 26:2029-2035.

Wang N, Erickson RJ, Ingersoll CG, Ivey CD, Brunson EL, Augspurger T, Barnhart MC. 2008.

Influence of pH on the acute toxicity of ammonia to juvenile freshwater mussels (fatmucket,

Lampsilis siliquoidea). Environ Toxicol Chem 27:1141-1146.

Wang N, Mebane CA, Kunz JL, Ingersoll CG, May TW, Arnold WR, Santore RC, Augspurger

T, Dwyer FJ, Barnhart MC. 2009. Evaluation of acute copper toxicity to juvenile freshwater

mussels (fatmucket, Lampsilis siliquoidea) in natural and reconstituted waters. Environ Toxicol

Chem 28:2367-2377.

Wang N, Ingersoll CG, Ivey CD, Hardesty DK, May TW, Augspurger T, Roberts AD, Genderen

EV, Barnhart MC. 2010. Sensitivity of early life stages of freshwater mussels (Unionidae) to

acute and chronic toxicity of lead, cadmium, and zinc in water. Environ Toxicol Chem 29:2053-

2063.

Wang N, Consbrock R, Ingersoll CG, Barnhart MC. 2011a. Evaluation of influence of sediment

on the sensitivity of a unionid mussel (Lampsilis siliquoidea) to ammonia in 28-day water

exposures. Environ Toxicol Chem 30:2270-2276.

Wang N, Mebane CA, Kunz JL, Ingersoll CG, Brumbaugh WG, Santore RC, Gorsuch JW,

Arnold WR. 2011b. Influence of dissolved organic carbon on toxicity of copper to a unionid

mussel (Villosa iris) and a cladoceran (Ceriodaphnia dubia) in acute and chronic water

exposures. Environ Toxicol Chem 30:2115-2125.

Wang N, Ingersoll CG, Ivey CD, Hammer E, Bauer CR, Augspurger T, Raimondo S, Shephard

B, Bartoszek J, Barnhart C, Eckert N. 2012. Acute sensitivity of freshwater mollusks to select

chemicals with various toxic modes of action. Poster RP 200 presented at the 33rd Annual

meeting of the Society of Environmental Toxicology and Chemistry, November 15, Long Beach,

CA.

29

Wang N, Ingersoll CG, Ivey CD, Brumbaugh WG, Besser JM, Hammer E, Bauer CR,

Augspurger T, Raimondo S, Shephard B, Bartoszek J, Barnhart C, Eckert N. 2013. Acute

sensitivity of freshwater mollusks and commonly tested invertebrates to select chemicals with

different toxic modes of action. Poster MP030, Society of Environmental Toxicology and

Chemistry annual meeting, November 18, Nashville, TN.

30

Appendix

Individual toxicity test results and supporting test water chemistry for whole effluent tests

conducted in 2010 and 2011 (Tables A1 to A4), mock effluent tests conducted in 2012

(Tables B1 to B3), and reference toxicant tests conducted between 2010 and 2012 (Tables

C1 to C8)

31

Table A1. Water quality characteristics of two North Carolina permitted effluent samples measured at the beginning and the end of 7-d tests conducted in 2010 with cladoceran (Ceriodaphnia dubia [CD]), fathead minnow (Pimephales promelas [FHM]), and freshwater mussels (notched rainbow [NR], Villosa constricta and yellow lance [YL], Elliptio lanceolata).

Test day

Test species

Dissolved oxygen (mg/L)

pH Conductivity Hardness Alkalinity Ammonia (mg N/L)

Major cations and anions (mg/L)

Effluent Dilution (µS/cm) (mg/L as CaCO3)

Ca2+

K+ Mg

2+ Na

+ SO4

2- Cl

-

Effluent 1

0 0% All 8.3 8.27 247 100 86 0.04 0 6.25% All 8.2 8.26 257 96 84 0.05 0 12.5% All 8.3 8.22 278 92 80 0.06

0 25% All 8.3 8.17 303 86 80 0.06

0 50% All 8.5 8.00 363 70 68 0.01

0 100% All 8.6 7.65 496 40 50 0.16

13.2 3.4 87.8 9.4 74.8 51.0

2 100%a All 9.0 7.84 485 40 50 0.43

Calculated hardness of 100% effluent sample based on measured Mg and Ca:

7 0% NR 7.1 8.20 265 106 94 0.19

7 25% NR 6.9 8.08 315 90 84 0.11

399 (mg/L as CaCO3)

7 100% NR 6.9 7.90 502 44 64 0.30

The colorometric measure of hardness is prone to interferences (e.g., elevated Fe can cause an artifact).

7 0% YL 7.1 8.16 269 110 100 0.16

7 25% YL 6.9 8.06 324 102 100 0.16

7 100% YL 7.0 7.90 503 50 64 0.26

7 0% FHM 7.1 8.12 336 114 94 0.19

7 25% FHM 7.5 7.90 366 100 86 0.05

7 100% FHM 7.1 7.89 572 48 62 0.20

7 0% CD 7.6 8.44 270 112 100 0.19

7 25% CD 7.7 8.31 340 100 90 0.21

7 100% CD 7.6 8.10 544 50 64 0.55

Continued on next page

a The second batch of effluent samples were also measured when receiving on test Day 2.

b Red numbers indicate ammonia concentrations above EPA chronic water quality criterion (USEPA 2013).

c Bold red numbers indicates ammonia concentrations above EPA acute water quality criterion (USEPA 2013).

32

Table A1 (concluded). Water quality characteristics of two North Carolina permitted effluent samples measured at the beginning and the end of 7-d tests conducted in 2010 with cladoceran (Ceriodaphnia dubia [CD]), fathead minnow (Pimephales promelas [FHM]), and freshwater mussels (notched rainbow [NR], Villosa constricta and yellow lance [YL], Elliptio lanceolata).

Test day

Test species

Dissolved oxygen (mg/L)

pH Conductivity Hardness Alkalinity Ammonia (mg N/L)

Major cations and anions (mg/L)

Effluent Dilution (µS/cm) (mg/L as CaCO3)

Ca2+

K+ Mg

2+ Na

+ SO4

2- Cl

-

Effluent 2

0 0% (the same control for Effluent 1 test)

0 6.25% All 8.1 8.39 248 98 74 0.22 0 12.5% All 8.3 8.32 246 90 80 0.41b

0 25% All 8.2 8.26 248 78 80 0.72

0 50% All 8.4 8.12 254 60 80 1.78

0 100% All 8.4 7.87 273 18 56 2.71

5.4 1.2 65.4 5.6 13.6 40.8

2 100%a All 9.0 7.99 374 20 84 3.41c

Calculated hardness of 100% effluent sample based on measured Mg and Ca:

7 25% NR 7.1 8.12 279 84 90 0.65

7 100% NR 6.0 7.68 338 18 68 0.91

286 (mg/L as CaCO3)

7 25% YL 7.0 8.07 278 80 88 0.63

7 100% YL 6.0 7.58 338 16 70 1.33

7 0% FHM 7.1 8.12 360 114 94 0.14

7 25% FHM 7.1 8.11 332 88 90 0.63

7 100% FHM 7.0 8.00 415 18 80 2.78

7 0% CD 7.8 8.46 297 112 92 0.29

7 25% CD 7.6 8.39 303 90 90 0.61

7 100% CD 7.5 8.20 367 20 78 1.91 a The second batch of effluent samples were also measured when receiving on test Day 2.

b Red numbers indicate ammonia concentrations above EPA chronic water quality criterion (USEPA 2013). c Bold red numbers indicates ammonia concentrations above EPA acute water quality criterion (USEPA 2013).

33

Table A2. Responses of cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and two freshwater mussels (notched rainbow, Villosa constricta; yellow lance, Elliptio lanceolata) in 7-d exposures to two effluent samples. Values are means with standard deviation in parentheses (n = 10 for the cladoceran and 4 for other three species). Value in the shaded cells indicate a significant reduction in an endpoint relative to control (Dunnett's test or Steel's many-one rank test, p< 0.05; USEPA 2002). No-observed-effect concentration [NOEC], lowest-observed-effect concentration [LOEC]), and 20% inhibition concentrations (IC20) with 95% confidence interval (CI) are presented for each endpoint. Mussel length data at the concentration above the NOEC for survival (means below dashed lines) were excluded from hypothesis testing when calculating the NOEC and LOEC

Cladoceran

Fathead minnow

Notched rainbow Yellow lance

Treatment Survival (%) No. of young Survival (%) Biomass (mg) Survival (%) Length (mm)a Survival (%) Length (mm)

Effluent 1

0% 90 22 (8.9)

100 (0) 3.83 (0.37)

92 (10) 0.40 (0.03)

51 (30) NMb

6.25% 80 14 (8.1)

100 (0) 3.30 (0.40)

100 (0) 0.47 (0.03)

45 (52) NM

12.5% 90 15 (7.0)

100 (0) 3.78 (0.82)

93 (5.0) 0.37 (0.04)

20 (23) NM

25% 100 16 (4.9)

95 (5.8) 3.15 (0.62)

70 (41) 0.41 (0.03)

40 (32) NM

50% 100 16 (6.2)

98 (5.0) 3.48 (0.52)

93 (15) 0.42 (0.02)

93 (5.0) NM

100% 100 17 (5.9) 98 (5.0) 3.63 (0.42) 93 (15) 0.46 (0.04) 73 (35) NM

NOEC (%) 100 100 100 100

100 100

100c

LOEC (%) >100 >100

>100 >100

>100 >100

>100c

IC20 (CI; %) >100 <6.25 >100 >100 >100 >100 >100

c

Effluent 2

0% 90 21 (8.6)

100 (0) 4.38 (0.28)

92 (10) 0.40 (0.03)

51 (30) NM

6.25% 90 20 (7.1)

100 (0) 4.10 (0.50)

98 (5.0) 0.43 (0.02)

48 (36) NM

12.5% 100 26 (9.3)

98 (5.0) 4.30 (0.25)

90 (14) 0.39 (0.05)

57 (40) NM

25% 100 28 (4.1)

100 (0) 4.78 (0.34)

65 (36) 0.41 (0.02)

67 (38) NM

50% 100 30 (3.9)

100 (0) 3.93 (0.39)

85 (13) 0.34 (0.01)

48 (37) NM

100% 90 31 (8.5) 100 (0) 3.60 (0.22) 38 (43) 0.35 (0.03)d 2.5 (5.0) NM

NOEC (%) 100 100 100 50 50 25

50c

LOEC (%) >100 >100

>100 100

100 50

100c

IC20 (CI; %) >100 >100 >100 >100 24 (12-67) >100 53 (42-67)

c

a Mean initial shell length and standard deviation at the beginning of the test: 0.284 ± 0.029 mm (n = 14).

b Not measured because of low control survival.

c The value should be used with caution due to low control survival.

d Based on only three replicates (n=3) due to 100% mortality in one replicate at this exposure concentration.

34

Table A3 Water quality characteristics of three North Carolina effluent samples measured at the beginning and the end of 7-d test conducted in 2011 with cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas [FHM]), and freshwater mussel (notched rainbow [NR], Villosa constricta).

Test day

Test species

Dissolved oxygen (mg/L)

pH Conductivity Hardness Alkalinity Ammonia (mg N/L)

Major cations and anions (mg/L)

Effluent Dilution (µS/cm) (as CaCO3)

Ca2+

K+ Mg

2+ Na

+ NO3

- SO4

2- Cl

-

Effluent 3

0 0% All 8.4 7.99 257 100 90 0.07 0 25% All 9.5 7.95 400 100 92 0.02 0 100% All 10.5 7.53 417 50 82 0.04

15.0 11.5 3.5 69.1 2.6 18.3 66.9 2 100%a All 10.3 7.69 485 50 100 0.03

7 0% NR 7.3 8.09 247 100 90 0.05

7 25% NR 7.4 8.04 301 90 90 0.03

7 100% NR 7.4 8.07 466 60 88 0.02

7 0% FHM 7.0 8.03 384 112 100 0.55

7 25% FHM 6.8 7.96 395 100 92 0.19

7 100% FHM 7.0 8.06 599 62 100 0.17

Effluent 4

0 0% (the same control for Effluent 3 test) 0 25% All 9.5 8.05 358 104 90 0.04 0 100% All 11.5 7.33 657 120 94 0.08

45.3 13.9 5.2 80.4 98.9 41 60.5

2 100%a All 10.3 7.69 485 112 100 0.06

7 0% NR 7.2 8.12 250 100 90 0.05

7 25% NR 6.9 8.07 348 104 90 0.02

7 100% NR 6.8 8.10 649 120 106 0.02

7 0% FHM 7.0 8.03 389 118 100 0.90

7 25% FHM 6.8 8.02 467 116 100 0.22

7 100% FHM 6.6 8.02 765 118 110 0.15

Effluent 5

0 0% (the same control for Effluent 3 test) 0 25% All 9.6 8.16 485 76 82 0.09 0 100% All 10.7 7.27 594 38 70 0.16

12.2 10.5 3.4 105 53.7 58.9 67.7

2 100%a All 9.9 7.35 572 46 64 0.13

7 0% NR 6.9 8.12 244 92 90 0.07

7 25% NR 6.9 8.04 333 82 82 0.07

7 100% NR 6.9 7.90 606 56 76

7 0% FHM 7.3 8.20 362 102 92 0.55

7 25% FHM 7.0 8.26 490 104 100 0.13

7 100% FHM 6.7 7.87 681 60 70 0.05

a The second batch of effluent samples were also measured when receiving on test Day 2.

35

Table A4. Responses of cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and notched rainbow (Villosa constricta) in 7-d exposures to three permitted effluent samples. Values are means with standard deviation in parentheses (n=10 for the cladoceran and n=4 for other two species). Values in the shaded cell indicates a significant reduction in an endpoint relative to control (p< 0.05). No-observed-effect concentration [NOEC] and the lowest-observed-effect concentration [LOEC]), and 20% inhibition concentrations (IC20) with 95% confidence interval (CI) are presented for each endpoint. Mussel length data at the concentration above the NOEC for survival (means below dashed lines) were excluded from hypothesis testing when calculating the NOEC and LOEC.

Cladoceran

Fathead minnow

Notched rainbow

Treatment Survival

(%) No. of young

Survival (%) Biomass (mg) Survival

(%) Length (mm)

a

Effluent 3

0% 30 9 (8.7)

88 (13) 5.00 (1.22)

93 (9.6) 0.40 (0.03)

6.25% 40 3 (4.0)

93 (10) 5.40 (1.15)

69 (21) 0.38 (0.02)

12.5% 20 2 (2.9)

95 (5.7) 5.20 (0.67)

63 (15) 0.33 (0.04)

25% 30 2 (3.7)

95 (10) 5.05 (0.70)

59 (22) 0.37 (0.04)

50% 50 0.5 (1.6)

85 (10) 4.85 (0.58)

72 (13) 0.36 (0.09)

100% 40 0.8 (2.5) 95 (5.8) 5.90 (0.54) 65 (5.8) 0.40 (0.05)

NOEC (%) NRb NR 100 100 50 50

LOEC (%) NR NR

>100 >100

100 >50

IC20 (CI; %) NR NR >100 >100 <6.25 >100

Effluent 4

0% 60 12 (11)

85 (10) 4.62 (0.97)

93 (9.6) 0.40 (0.01)

6.25% 70 6 (8.8)

95 (10) 6.32 (0.38)

85 (10) 0.39 (0.03)

12.5% 70 8 (11)

95 (5.8) 6.50 (0.82)

85 (10) 0.37 (0.04)

25% 70 14 (11)

100 (0) 6.40 (0.36)

37 (20) 0.40 (0.03)

50% 60 15 (17)

98 (5.0) 6.73 (0.42)

41 (7.0) 0.36 (0.06)

100% 50 15 (16) 95 (5.8) 7.40 (0.62) 18 (24) 0.38 (0.01)c

NOEC NR NR 100 100 12.5 12.5

LOEC NR NR

>100 >100

25 >12.5

IC20 (CI) NR NR >100 >100 69 (23-120) >100

Effluent 5

0% 100 15 (5.6)

85 (10) 6.98 (0.17)

91 (11) 0.42 (0.03)

6.25% 30 3 (5.5)

98 (5.0) 8.02 (0.61)

77 (16) 0.43 (0.05)

12.5% 40 1 (4.4)

98 (5.0) 6.30 (1.60)

73 (15) 0.42 (0.01)

25% 40 0.8 (1.6)

100 (0) 7.27 (0.36)

83 (21) 0.39 (0.02)

50% 70 4 (5.0)

98 (5.0) 7.77 (0.43)

85 (5.8) 0.40 (0.01)

100% 40 1 (3.0) 95 (5.8) 8.12 (0.42) 88 (19) 0.37 (0.05)

NOEC NR NR 100 100 100 100

LOEC NR NR

>100 >100

>100 >100

IC20 (CI) NR NR >100 >100 >100 >100 a Mean initial shell length and standard deviation at the beginning of the test: 0.330 ± 0.059 mm (n = 19).

b Not reported due to low control survival in two of the three effluent tests (possible due to low quality of test

organisms used).

c Based on only two replicates (n=2) due to 100% mortality in two replicates at this exposure concentration.

36

Table B1. Concentrations of ammonia and five metals in two mock effluents used in 7-d toxicity tests with cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and notched rainbow (Villosa constrict). Standard deviation in parentheses (n = 1 to 2 for metals and 7 for ammonia)

Species Mock

effluent Dilution

Total ammonia (mg N/L)

Nickel (µg/L)

Copper (µg/L)

Zinc (µg/L)

Cadmium (µg/L)

Lead (µg/L)

Nominal Measured Nominal Measured Nominal Measured Nominal Measured Nominal Measured Nominal Measured Cladoceran 1 0% 0 0.30 (0.32)

0 0.9 (0.2)

0 0.4 (0.2)

0 7.6 (1.6)

0 0.02 (0.01)

0 0.05 (0.04)

3.12% 0.25 0.32 (0.15)

13 13 (0.8)

0.4 0.7 (0.1)

30 26 (2.9)

0.06 0.06 (0.02)

0.3 0.14 (0.0)

6.25% 0.5 0.54 (0.14)

26 26 (1)

0.8 1.1 (0.1)

60 47 (6.5)

0.13 0.11 (0.04)

0.6 0.25 (0.1)

12.5% 1.0 0.99 (0.10)

52 55

1.5 1.6

120 96

0.25 0.23

1.3 0.5

25% 2.0 1.98 (0.14)

104 109

3.0 2.5

240 195

0.5 0.46

2.5 1.0

50% 4.0 4.01 (0.15)

208 217

6.0 5.4

480 464

1.0 1.03

5 3.2

2 0% 0 0.24 (0.22)

0 0.9 (0.2)

0 0.4 (0.2)

0 7.6 (1.6)

0 0.02 (0.01)

0 0.05 (0.04)

3.12% 0.1 0.16 (0.08)

12 12 (0.6)

2 2.0 (0.3)

31 27 (2.3)

2 1.7 (0.2)

6 2.7 (0.2)

6.25% 0.2 0.26 (0.13)

23 24 (2)

4 3.0 (0.5)

62 41 (11)

4 3.1 (0.9)

11 4.4 (1.0)

12.5% 0.4 0.42 (0.04)

46 49

8 6.0

124 95

8 8.0

22 10

25% 0.8 0.80 (0.06)

92 97

16 13

248 232

16 16

44 28

50% 1.6 1.63 (0.17) 184 193 32 26 496 483 32 33 88 62

Fathead minnow 1 0% 0 0.14 (0.09)

0 0.9 (0.2)

0 0.3 (0.03)

0 7.0 (0.6)

0 0.01 (0.0)

0 0.05 (0.4)

6.25% 0.5 0.35 (0.48)

26 31 (5.3)

0.8 1.0 (0.02)

60 59 (24)

0.13 0.15 (0.02)

0.6 0.28 (0.1)

12.5% 1.0 0.60 (0.91)

52 60 (7.2)

1.5 1.7 (0.1)

120 119 (33)

0.25 0.25 (0.03)

1.3 0.63 (0.2)

25% 2.0 1.88 (0.34)

104 120 (16)

3.0 2.7 (0.4)

240 239 (62)

0.5 0.53 (0.09)

2.5 1.4 (0.6)

50% 4.0 3.81 (0.60)

208 239 (31)

6.0 5.5 (0.2)

480 520 (79)

1.0 1.1 (0.12)

5 3.7 (0.7)

100% 8.0 8.29 (0.27)

416 444

12 10

960 951

2.0 2.0

10 8

2 0% 0 0.18 (0.14)

0 0.8 (0.0)

0 0.2 (0)

0 6.5 (0.0)

0 0.02 (0.01)

0 0.1 (0.0)

6.25% 0.2 0.22 (0.04)

23 27 (2.6)

4 3.3 (1)

62 53 (27)

4 4.1 (0.5)

11 5.6 (2.9)

12.5% 0.4 0.42 (0.04)

46 53 (6.5)

8 6.9 (1)

124 122 (38)

8 8.6 (0.9)

22 14 (5.7)

25% 0.8 0.77 (0.09)

92 109 (17)

16 14 (1)

248 274 (59)

16 18 (2.6)

44 36 (11)

50% 1.6 1.53 (0.32)

184 214 (30)

32 27 (3)

496 547 (90)

32 36 (4.8)

88 76 (20)

100% 3.2 3.26 (0.12) 368 383 64 54 992 1040 64 66 176 158

Notched rainbow 1 0% 0 0.07 (0.01)

0 0.9 (0.1)

0 0.2 (0.1)

0 6.8 (0.4)

0 0.014 (0.0)

0 0.05 (0.03)

3.12% 0.25 0.27 (0.04)

13 14 (0.4)

0.4 0.5 (0.2)

30 30 (8.7)

0.06 0.07 (0.0)

0.3 0.2 (0.0)

6.25% 0.5 0.48 (0.07)

26 27 (0.0)

0.8 0.8 (0.3)

60 53 (15)

0.13 0.14 (0.01)

0.6 0.3 (0.1)

12.5% 1.0 0.93 (0.09)

52 55 (0.5)

1.5 1.4 (0.4)

120 111 (21)

0.25 0.25 (0.03)

1.3 0.6 (0.1)

25% 2.0 1.93 (0.21)

104 110 (1.4)

3.0 2.4 (0.1)

240 223 (38)

0.5 0.49 (0.04)

2.5 1.3 (0.4)

50% 4.0 3.91 (0.32)

208 219 (2.1)

6.0 4.9 (0.6)

480 478 (20)

1.0 1.0 (0.02)

5 3.4 (0.3)

100% 8.0 8.57 (0.27)

416 444 (0.7)

12 10 (0.1)

960 947 (6.4)

2.0 2.0 (0.06)

10 8.0 (0.1)

2 0% 0 0.07 (0.01)

0 0.88 (0.2)

0 0.2 (0.1)

0 6.8 (0.4)

0 0.01 (0.01)

0 0.05 (0.04)

3.12% 0.1 0.13 (0.02)

12 13 (0.1)

2 1.6 (0.1)

31 30 (6.0)

2 1.9 (0.1)

6 2.8 (0.1)

6.25% 0.2 0.21 (0.02)

23 25 (0.1)

4 2.8 (0.01)

62 47 (20)

4 3.9 (0.2)

11 4.5 (1.4)

12.5% 0.4 0.40 (0.06)

46 49 (0.4)

8 5.8 (0.2)

124 114 (28)

8 8.2 (0.3)

22 13 (4.1)

25% 0.8 0.77 (0.10)

92 97 (0.1)

16 13 (0.1)

248 250 (25)

16 17 (0.4)

44 33 (7.1)

50% 1.6 1.57 (0.22)

184 194 (0.7)

32 25 (0.5)

496 508 (35)

32 33 (0.9)

88 69 (10)

100% 3.2 3.26 (0.12)

368 383 (0.0)

64 53 (1.3)

992 1030 (14)

64 66 (0.8)

176 155 (4.9)

37

Table B2. Water quality characteristics of two mock effluents used in 7-d toxicity tests with cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and notched rainbow (Villosa constrict). Standard deviation in parentheses if n=2.

Species Mock

effluent Dilution

Dissolved oxygen (mg/L)

pH Conductivity Hardness Alkalinity

(µS/cm) (mg/L as CaCO3)

Cladoceran 1 0% 8.5 (0.3) 8.4 (0.02) 297 (47) 103 (1) 103 (4)

3.12% 8.6 (0.3) 8.4 (0.04) 273 (9) 103 (1) 100 (0)

6.25% 8.4 8.4 280 102 100

12.5% NM

a NM NM NM NM

25% 9.2 8.3 285 104 104

50% NM NM NM NM NM

2 0% 8.7 (0.1) 8.4 (0.03) 272 (11) 104 (3) 103 (4)

3.12% 8.6 (0) 8.4 (0.03) 276 (6) 105 (1) 99 (1)

6.25% 8.4 8.4 284 106 96

12.5% NM NM NM NM NM

25% 9.0 8.3 276 106 100

50% NM NM NM NM NM

Fathead minnow 1 0% 8.6 (0.2) 8.4 (0.02) 301 (52) 117 (21) 108 (3)

6.25% 8.6 NM 345 NM NM

12.5% 8.5 8.4 328 134 112

25% 8.9 (0.4) 8.3 313 (39) 104 104

50% 8.7 8.3 352 106 100

100% NM NM NM NM NM

2 0% 8.7 (0.2) 8.4 (0.02) 295 (43) 118 (22) 111 (7)

6.25% 8.4 NM 325 NM NM

12.5% 8.5 8.4 329 134 116

25% 8.7 (0.4) 8.3 300 (33) 106 100

50% 8.5 8.4 341 134 110

100% 10 8.3 286 106 100

Notched rainbow 1 0% 8.7 (0.1) 8.4 (0.1) 273 (12) 104 (3) 103 (4)

3.12% 8.7 (0.1) 8.3 274 (11) 102 100

6.25% 8.7 NM 284 NM NM

12.5% 8.8 8.4 288 106 102

25% 8.9 (0.2) NM 292 (9) 104 104

50% 9 8.3 318 106 100

100% 10.7 8.2 334 108 106

2 0% 9.0 (0.3) 8.4 (0.1) 273 (13) 103 (4) 103 (1)

3.12% 8.7 (0.2) 8.4 277 (6) 100 106

6.25% 9 NM 283 NM NM

12.5% 8.8 8.4 284 106 98

25% 8.9 (0.1) NM 283 (10) NM NM

50% 8.5 8.4 298 112 100

100% 10.8 8.3 288 106 100

a Not measured.

38

Table B3. Responses of cladoceran (Ceriodaphnia dubia), fathead minnow (Pimephales promelas), and notched rainbow (Villosa constricta) in 7-d exposures to two mock effluents. Values are means with standard deviation in parentheses (n=10 for the cladoceran and n=4 for other two species). Value in shaded cells indicate a significant reduction in an endpoint relative to control (p< 0.05). No-observed-effect concentration [NOEC], lowest-observed-effect concentration [LOEC]), and 20% inhibition concentrations (IC20) with 95% confidence interval (CI) are presented for each endpoint.

Cladoceran

Fathead minnow

Notched rainbow

Treatment Survival (%) No. of young Survival (%) Biomass (mg) Survival (%) Length (mm)a

Mock Effluent 1

0% 100 20 (6.3)

100 (0) 5.72 (0.72)

100 (0) 0.478 (0.023)

3.125% 100 15 (5.2)

NTb NT

100 (0) 0.410 (0.007)

6.25% 90 11 (4.4)

100 (0) 5.92 (0.79)

98 (5.0) 0.374 (0.005)

12.5% 10 1.0 (2.1)

100 (0) 6.18 (0.83)

90 (9.0) 0.358 (0.013)

25% 0 0

90 (20) 5.30 (1.16)

80 (18) 0.347 (0.005)

50% 0 0

60 (14) 3.58 (0.81)

0 NAb

100% NTc NT 0 0 NT NT

NOEC (%) 6.25 3.125

25 25

25 <3.125

LOEC (%) 12.5 6.25

50 50

50 3.125

IC20 (CI; %) 7.0 (4.3-7.8) <3.1 34 (21-41) 33 (20-40) 25 (15-28) 5.6 (4.2-10)

Mock Effluent 2

0% 100 21 (9.9)

100 (0) 6.50 (0.49)

100 (0) 0.473 (0.024)

3.125% 100 13 (6.4)

NT NT

97 (6.0) 0.428 (0.008)

6.25% 90 11 (4.4)

98 (5) 5.75 (0.53)

100 (0) 0.393 (0.011)

12.5% 0 0

93 (10) 6.70 (0.43)

95 (5.8) 0.350 (0.006)

25% 0 0

85 (17) 4.50 (1.52)

85 (13) 0.340 (0.009)

50% 0 0

50 (22) 2.02 (0.90)

0 NA

100% NT NT 0 0 NT NT

NOEC (%) 6.25 3.125 25 12.5 25 <3.125

LOEC (%) 12.5 6.25

50 25

50 3.125

IC20 (CI; %) 6.9 (4.3-7.5) <3.2 29 (19-36) 20 (16-28) 26 (21-29) 8.3 (5.7-11) a Mean initial shell length and standard deviation at the beginning of the test: 0.349 ± 0.025 mm (n = 20).

b Not applicable due to no survival at this exposure concentration; No length value at this concentration was used for calculating LOEC or IC20.

c Species was not tested at this exposure concentration.

39

Table C1. Survival and EC50s (95% confidence interval) in acute 4-d NaCl reference toxicant tests with two mussel species (notched rainbow, Villosa constricta and yellow lance, Elliptio lanceolata) in ASTM hard water (ASTM 2013a). Tests conducted in 2010.

Nominal concentration (g NaCl/L)

Measured salinity (g/L)

Survival (%)

Notched rainbow Yellow lance

0 0.0 100

100

1 1.2 100

93

2 2.2 100

60

4 4.4 53

0

8 8.6 0

0

16 16.7 0

0

EC50 (CI) 3.9 (3.3-4.7) 2.1 (1.8-2.5)

Note: Only 3 replicates per concentration due to limited mussels.

40

Table C2. Mean concentrations of copper (n=2; standard deviation in parentheses) and water quality characteristics in acute 4-d copper reference toxicity tests with three mussel species (notched rainbow, Villosa constricta; yellow lance, Elliptio lanceolata; and yellow lampmussel, Lampsilis cariosa). Tests conducted in 2011.

Species Nominal copper

(µg Cu/L)

Measured copper

(µg Cu/L)

Dissolved oxygen (mg/L)

pH Conductivity

(µS/cm)

Alkalinity Hardness

(mg/L as CaCO3)

Notched rainbow 0 0.8 (0.1) 7.69 8.3 567 120 169

6.25 5.3 (0.4) NM

a NM NM NM NM

12.5 10 (0.9) NM NM NM NM NM

25 19 (1.4) 7.66 8.3 564 120 172

50 37 (4.7) NM NM NM NM NM

100 70 (7.8) 7.74 8.4 560 120 166

Yellow lance 0 0.6 (0.1) 8.20 8.4 578 122 166

6.25 5.3 (0.5) NM NM NM NM NM

12.5 9.3 (0.9) NM NM NM NM NM

25 19 (1.5) 7.96 8.3 570 120 164

50 36 (4.0)

NM NM NM NM

100 70 (8.7) 8.12 8.3 570 120 166

Yellow lampmussel 0 0.7 (0.3) 8.10 8.4 552 114 156

6.25 5.2 (0.2) 7.97 8.4 522 114 148

12.5 10 (0.4) NM NM NM NM NM

25 20 (0.6) 7.75 8.4 528 114 156

50 39 (1.1) NM NM NM NM NM

100 75 (2.8) 8.10 8.4 552 114 156 a Not measured.

41

Table C3. Mean concentrations of total ammonia (n=4; standard deviation in parentheses) and water quality characteristics in acute 4-day ammonia reference toxicity tests with three mussel species (notched rainbow, Villosa constricta; yellow lance, Elliptio lanceolata; and yellow lampmussel, Lampsilis cariosa). Tests conducted in 2011.

Species Nominal ammonia (mg N/L)

Measured ammonia (mg N/L)

Dissolved oxygen (mg/L)

pH Conductivity

(µS/cm)

Alkalinity Hardness

(mg/L as CaCO3)

Notched rainbow 0 0.1 (0.1) 7.88 8.4 569 120 166

1 1.0 (0.1) NM

a 8.4 NM NM NM

2 2.0 (0.1) NM 8.4 NM NM NM

4 3.9 (0.1) 7.91 8.4 608 120 168

8 6.8 (0.5) NM 8.4 NM NM NM

16 18 (2.7) 7.91 8.3 731 120 168

Yellow lance 0 0.1 (0.01) 7.95 8.4 575 122 170

1 1.0 (0.1) NM 8.4 586 NM NM

2 1.9 (0.1) NM 8.3 594 NM NM

4 3.4 (0.2) 8.04 8.4 608 124 170

8 6.2 (0.1) NM 8.3 633 NM NM

16 17.4 (0.2) 7.87 8.3 731 128 170

Yellow lampmussel 0 0.1 (0.03) 7.86 8.4 538 110 144

1 1.1 (0.5) NM 8.4 NM NM NM

2 2.0 (0.7) NM 8.5 NM NM NM

4 3.6 (0.9) 7.75 8.5 586 114 136

8 7.0 (0.5) NM 8.4 NM NM NM

16 16 (1.6) 7.87 8.4 701 114 136 a Not measured.

42

Table C4. Mean salinity (n=2; standard deviation in parentheses) and water quality characteristics in acute 4-day NaCl reference toxicity test with notched rainbow (Villosa constricta). Tests conducted in 2011.

Species Nominal

NaCl (g/L)

Measured Salinity

(g/L)

Dissolved oxygen (mg/L)

pH Conductivity

(µS/cm)

Alkalinity Hardness

(mg/L as CaCO3)

Notched rainbow 0 0 (0) 8.8 8.4 569 120 166

1 1.1 (0.1) NM

a 8.4 NM NM NM

2 2.1 (0.0) NM 8.4 NM NM NM

4 4.2 (0.1) 8.81 8.4 608 120 168

8 8.2 (0.1) NM 8.4 NM NM NM

16 16.3 (0.1) 8.89 8.3 731 120 168

a Not measured.

43

Table C5. Survival and EC50s (95% confidence interval) in acute 4-d reference toxicant tests with three mussel species (notched rainbow, Villosa constricta; yellow lance, Elliptio lanceolata; and yellow lampmussel, Lampsilis cariosa) in ASTM hard water (ASTM 2013a). Tests conducted in 2011.

Notched rainbow

Yellow lance

Yellow lampmussel

Measured concentration

Survival (%)

Measured

concentration Survival

(%)

Measured concentration

Survival (%)

Copper (µg Cu/L)

0.78 100

0.56 100

0.67 100

5.34 90

5.26 100

5.24 100

9.97 50

9.32 75

10.45 80

18.80 40

18.55 25

20.35 90

36.85 0

35.75 24

39.30 40

70.30 0 70.15 5 74.50 42

EC50 (CI) 12 (9.6-15) 16 (13-20) 46 (32-68)

Total ammonia (mg N/L)

0.05 100

0.09 76

0.07 95

1.04 91

0.97 90

1.11 100

1.98 94

1.85 71

2.01 97

3.90 67

3.42 21

3.61 100

6.84 0

6.15 0

6.99 28

18.40 0

17.35 0

16.05 0

EC50 (CI) 3.5 (2.9-4.3) 2.2 (1.9-2.5) 8.2 (7.2-9.2)

Sodium chloride (salinity, g/L)

0.0 95

NTa

NT

1.1 100

NT

NT 2.1 100

NT

NT

4.2 53

NT

NT 8.2 0

NT

NT

16.3 0

NT

NT

EC50 (CI) 4.1 (3.5-4.8)a NT NT

a Not tested b EC50 for NaCl was estimated based on nominal NaCl concentrations of 0, 1, 2, 4, 8, and 16 g NaCl/L.

44

Table C6. Mean copper concentrations and water quality characteristics (n=2; standard deviation in parentheses) in acute 4-day copper reference toxicity tests with three mussel species (yellow lance, Elliptio lanceolata; notched rainbow, Villosa constrict; Tar River spinymussel, Elliptio steinstansana). Tests conducted in 2012.

Species Nominal copper

(µg Cu/L)

Measured copper

(µg Cu/L)

Dissolved oxygen (mg/L)

pH Conductivity

(µS/cm)

Alkalinity Hardness

(mg/L as CaCO3)

Notched rainbow 0 1.1 8.7 8.6 588 124 154

6.25 7.2 NM

a NM NM NM NM

12.5 8.6 NM NM NM NM NM

25 25 8.6 8.6 589 116 148

50 33 NM NM NM NM NM

100 88 8.7 8.6 598 120 160

Yellow lance 0 0.8 (0.01) 8.4 (0.3) 8.5 (0) 602 (37) 119 (4) 167 (4)

6.25 5.8 (0.4) 8.3 (0.3) 8.3 (0) 610 (3) 120 (0) 163 (1)

12.5 11 (0.1) NM NM NM NM NM

25 23 (0.4) 8.5 (0.2) 8.4 (0.1) 586 (13) 117 (4) 164 (6)

50 44 (0.2) NM NM NM NM NM

100 81 (3.8) 8.4 (0.4) 8.4 (0.1) 581 (11) 117 (4) 159 (10)

Spinymussel 0 0.7 (0.04) 8.4 (0.4) 8.5 (0) 582 (8) 118 (3) 167 (4)

6.25 6.8 (0.3) 8.4 (0.4) 8.4 (0.01) 584 (6) 121 (1) 164 (3)

12.5 11 (0.6) NM NM NM NM NM

25 22 (1.1) 8.5 (0.3) 8.4 (0.02) 581 (6) 117 (4) 162 (2)

50 44 (0.4) NM NM NM NM NM

100 82 (7.2) 8.5 (0.4) 8.4 (0.1) 579 (8) 116 (3) 158 (8) a Not measured.

45

Table C7. Mean concentrations of total ammonia (n=4) and water quality characteristics (n=2) for acute 4-day ammonia toxicity tests with yellow lance (Elliptio lanceolata) and Tar River spinymussel (Elliptio steinstansana). Standard deviation in parentheses. Tests conducted in 2012.

Nominal ammonia (mg N/L)

Measured ammonia (mg N/L)

Dissolved oxygen (mg/L)

pH Conductivity

(µS/cm)

Alkalinity Hardness

(mg/L as CaCO3)

0 0.1 (0.02) 8.4 (0.1) 8.5 (0.03) 616 (16) 121 (4) 163 (4)

1 0.8 (0.12) 8.6 (0.1) 8.4 (0.3) 633 (30) 123 (3) 164 (3)

2 1.6 (0.18) NMa NM NM NM NM

4 3.6 (0.26) 8.4 (0.1) 8.4 (0.01) 659 (18) 122 (3) 164 (6)

8 7.7 (0.40) NM NM NM NM NM

16 18 (0.41) 8.2 (0.4) 8.3 (0.01) 729 (66) 123 (4) 166 (3)

a Not measured.

46

Table C8. Survival and EC50s (95% confidence interval, CI) in acute 4-d reference toxicant tests with three mussel species (yellow lance, Elliptio lanceolata; notched rainbow, Villosa constrict; Tar River spinymussel, Elliptio steinstansana). Tests conducted in 2012.

Notched rainbow

Yellow lance

Spinymussel

Measured concentration

Survival (%)

Measured

concentration Survival

(%)

Measured concentration

Survival (%)

Copper (µg Cu/L)

1.1 100

0.8 100

0.8 100

7.2 100

6.0 100

6.8 100

8.6 100 11 60

11 95

25 90

23 35

22 80

33 75

44 5

44 5

88 0 81 0 82 0

EC50 (CI) 39 (32-48) 16 (13-19) 26 (22-31)

Total ammonia (mg N/L)

NT

a

0.1 100

0.1 100

NT

0.8 100

0.8 96

NT

1.6 69

1.6 100

NT

3.6 61

3.6 54

NT

7.7 0

7.7 0

NT 18 0 18 0

EC50 (CI) 3.1 (2.6-3.7) 3.6 (3.1-4.3) a Not tested